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26.  Inhibiting autophagy by shRNA knockdown 
Autophagy  2013;9(10):1449-1450.
A glance through Autophagy or any other journal in this field shows that it is very common to block autophagy by RNA interference-based knockdown of ATG mRNAs in mammalian cell lines. Our lab’s experience is that this approach can easily make for failed experiments because good knockdown of even essential autophagy regulators does not necessarily mean you will get good inhibition of autophagy, and, over time, cells can find ways to circumvent the inhibitory effects of the knockdown.
PMCID: PMC3974882  PMID: 23800703
autophagy; shRNA; ATG5; knockdown; RNA interference
27.  Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes 
Autophagy  2009;5(8):1099-1106.
In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serum-containing Waymouth’s medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1α mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.
PMCID: PMC4170191  PMID: 19783904
cyclosporin A; dedifferentiation; mitochondrial permeability transition; mitophagy; mtDNA; remodeling
28.  Protector turns predator 
Autophagy  2013;9(9):1438-1439.
Therapy-induced autophagy is recognized as a critical determinant of treatment outcome in cancer patients, primarily as a factor underlying drug resistance. However, recent investigations point toward a context-dependent, death-inducing role for autophagy, the mechanism of which remains largely unknown. Our recent study provides evidence that autophagy can directly mediate cell killing in multiple tumor cell types by facilitating degradation of KRAS/K-Ras, a key survival protein. These findings have broad implications for strategies employing autophagy modulation to target tumor cells.
PMCID: PMC4026028  PMID: 24121562
autophagy; KRAS; tamoxifen; PRKC; EGFR; tumor cells
29.  TRP53 activates a global autophagy program to promote tumor suppression 
Autophagy  2013;9(9):1440-1442.
The mechanisms by which the TP53/TRP53 transcription factor acts as a tumor suppressor remain incompletely understood. To gain new insights into TP53/TRP53 biology, we used ChIP-seq and RNA-seq technologies to define global TRP53 transcriptional networks in primary cells subjected to DNA damage. Intriguingly, we identified a TRP53-regulated autophagy program, which can be coordinately regulated by the TRP53 family members TRP63 and TRP73 in certain settings. While autophagy is not involved in TRP53-dependent cell cycle arrest, it contributes to both TRP53-driven apoptosis in response to DNA damage and TRP53-mediated transformation suppression. Collectively, our genome-wide analyses reveal a profound role for TRP53 in regulating autophagy, through an extensive transcriptional network, and have demonstrated an important role for this program in promoting TRP53-mediated apoptosis and tumor suppression.
PMCID: PMC4026029  PMID: 23899499
p53; ChIP-seq; RNA-seq; tumor suppression; autophagy; apoptosis
30.  20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body 
Autophagy  2013;9(8):1172-1187.
Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.
PMCID: PMC3748190  PMID: 23674061
20-hydroxyecdysone; fat body; autophagy; Atg genes; Atg1; ATG8; transcriptional regulation; TORC1; Bombyx mori
31.  ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis 
Autophagy  2013;9(8):1188-1200.
The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.
PMCID: PMC3748191  PMID: 23782979
ATG4B; autophagin-1; autophagy; colitis; inflammation; intestinal homeostasis; cysteine peptidase; Paneth cell
32.  Drosophila Fip200 is an essential regulator of autophagy that attenuates both growth and aging 
Autophagy  2013;9(8):1201-1213.
Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1’s autophagy-, aging- and growth-regulating functions.
PMCID: PMC3748192  PMID: 23819996
autophagy; neurodegeneration; Drosophila; aging; growth
33.  IκB kinase complex (IKK) triggers detachment-induced autophagy in mammary epithelial cells independently of the PI3K-AKT-MTORC1 pathway 
Autophagy  2013;9(8):1214-1227.
Adherent cells require proper integrin-mediated extracellular matrix (ECM) engagement for growth and survival; normal cells deprived of proper ECM contact undergo anoikis. At the same time, autophagy is induced as a survival pathway in both fibroblasts and epithelial cells upon ECM detachment. Here, we further define the intracellular signals that mediate detachment-induced autophagy and uncover an important role for the IκB kinase (IKK) complex in the induction of autophagy in mammary epithelial cells (MECs) deprived of ECM contact. Whereas the PI3K-AKT-MTORC1 pathway activation potently inhibits autophagy in ECM-detached fibroblasts, enforced activation of this pathway is not sufficient to suppress detachment-induced autophagy in MECs. Instead, inhibition of IKK, as well as its upstream regulator, MAP3K7/TAK1, significantly attenuates detachment-induced autophagy in MECs. Furthermore, function-blocking experiments corroborate that both IKK activation and autophagy induction result from decreased ITGA3-ITGB1 (α3β1 integrin) function. Finally, we demonstrate that pharmacological IKK inhibition enhances anoikis and accelerates luminal apoptosis during acinar morphogenesis in three-dimensional culture. Based on these results, we propose that the IKK complex functions as a key mediator of detachment-induced autophagy and anoikis resistance in epithelial cells.
PMCID: PMC3748193  PMID: 23778976
autophagy; anoikis; extracellular matrix; integrin; mammary epithelial cells
34.  Autophagic degradation of leaf starch in plants 
Autophagy  2013;9(8):1247-1248.
Autophagy is an evolutionarily conserved process in eukaryotic cells that functions to degrade cytoplasmic components in the vacuole or lysosome. Previous research indicates that the core molecular machinery of autophagosome formation works well in plants, and plant autophagy plays roles in diverse biological processes such as nutrient recycling, development, immunity and responses to a variety of abiotic stresses. Recently, we reported that autophagy contributed to leaf starch degradation, which had been thought to be a process confined to chloroplasts. This finding demonstrated a previously unidentified pathway of leaf starch depletion and a new role of basal autophagy in plants.
PMCID: PMC3748196  PMID: 23722252
autophagy; ATG; leaf starch degradation; SSGL; stromule
35.  Tyrosine kinase inhibition facilitates autophagic SNCA/α-synuclein clearance 
Autophagy  2013;9(8):1249-1250.
The effects of ABL1/ABL inhibition on clearance of SNCA/α-synuclein were evaluated in animal models of α-synucleinopathies. Parkinson disease (PD) is a movement disorder characterized by death of dopaminergic substantia nigra (SN) neurons and brain accumulation of SNCA. The tyrosine kinase ABL1 is activated in several neurodegenerative diseases. An increase in ABL1 activity is detected in human postmortem PD brains. Lentiviral expression of SNCA in the mouse SN activates ABL1 via phosphorylation, while lentiviral Abl expression increases SNCA levels. Administration of the brain-penetrant tyrosine kinase inhibitor Nilotinib decreases Abl activity and facilitates autophagic clearance of SNCA in transgenic and lentiviral gene transfer models. Subcellular fractionation demonstrates accumulation of SNCA and hyperphosphorylated MAPT/Tau (p-MAPT) in autophagic vacuoles in SNCA-expressing brains, while Nilotinib treatment leads to protein deposition into the lysosomes, suggesting enhanced autophagic clearance. These data suggest that Nilotinib may be a therapeutic strategy to degrade SNCA in PD and other α-synucleinopathies.
PMCID: PMC3748197  PMID: 23787811
Nilotinib; Tau; autophagy; dopamine; α-synuclein
36.  A genetic model with specifically impaired autophagosome–lysosome fusion 
Autophagy  2013;9(8):1251-1252.
Yeast studies identified the evolutionarily conserved core ATG genes responsible for autophagosome formation. However, the SNARE-dependent machinery involved in autophagosome fusion with the vacuole in yeast is not conserved. We recently reported that the SNARE complex consisting of Syx17 (Syntaxin 17), ubisnap (SNAP-29) and Vamp7 is required for the fusion of autophagosomes with late endosomes and lysosomes in Drosophila. Syx17 mutant flies are viable but exhibit neuronal dysfunction, locomotion defects and premature death. These data point to the critical role of autophagosome clearance in organismal homeodynamics.
PMCID: PMC3748198  PMID: 23819962
autophagy; autophagosome; Drosophila; lysosome; neurodegeneration; SNARE; Syntaxin 17; ubisnap/SNAP-29; Vamp7
37.  Role of Epg5 in selective neurodegeneration and Vici syndrome 
Autophagy  2013;9(8):1258-1262.
Autophagy activity is essential for the survival of neural cells. Impairment of autophagy has been implicated in the pathogenesis of neurodegenerative disorders. Unlike the massive neuron loss in mice deficient for autophagy genes essential for autophagosome formation, we demonstrated that mice deficient for the metazoan-specific autophagy gene Epg5 develop selective neuronal damage and exhibit key characteristics of amyotrophic lateral sclerosis. Epg5 deficiency blocks the maturation of autophagosomes into degradative autolysosomes, slows endocytic degradation and also impairs endocytic recycling. Recessive mutations in human EPG5 have recently been causally associated with the multisystem disorder Vici syndrome. Here we show that while Epg5 knockout mice display some features of Vici syndrome, many phenotypes are absent.
PMCID: PMC3748201  PMID: 23674064
autophagy; autophagosome; Epg5; Vici syndrome; neurodegeneration
38.  β-adrenergic receptor-stimulated lipolysis requires the RAB7-mediated autolysosomal lipid degradation 
Autophagy  2013;9(8):1228-1243.
Hormone-stimulated lipolysis is a rapid way to mobilize fat from its storage depot for use in peripheral tissues. By convention, activation of cytosolic lipases via the β-adrenergic receptor (ADRB2)-cAMP signaling pathway is the only molecular mechanism considered to liberate fatty acids from triglycerides stored in lipid droplets (LDs) of cells. Herein, we provide evidence that, aside from the activation of cytosolic lipases, autophagy contributes to this hormone-stimulated lipolysis. The ADRB2-stimulated lipolysis was reduced after inhibition of early or late autophagy using either pharmacological inhibitors or shRNA-mediated autophagic gene knockdown. ADRB2 stimulation has caused a marked increase in the autophagy-targeted LDs for lysosomal degradation, which is dependent on the LD-associated RAB7 as evidenced by the use of both shRNA-mediated RAB7 knockdown and a dominant-negative RAB7 mutant. In addition, RAB7 is involved in unstimulated (basal) lipolysis, and mediates the enhanced basal lipolysis in PLIN1/perilipin 1 knockdown fat cells. In conclusion, our results showed a contribution of lipophagy to both basal and hormone-stimulated lipolysis and that RAB7 plays a pivotal role in the regulation of this autolysosome-mediated lipid degradation in fat cells.
PMCID: PMC3748194  PMID: 23708524
lipophagy; lipolysis; RAB7; autolysosome-mediated lipid degradation; 3T3-L1
39.  TFEB 
Autophagy  2013;9(8):1244-1246.
Parkinson disease (PD) is characterized by the progressive loss of nigral dopamine neurons and the presence of accumulations containing the disease-causing protein SNCA/α-synuclein. Here we review our recent findings describing how SNCA impairs the function of the master regulator of the autophagy-lysosomal pathway (ALP), the transcription factor EB (TFEB), and that genetic or pharmacological stimulation of its activity promotes protection of dopamine neurons. These findings suggest that strategies aimed at enhancing autophagy-mediated degradation of SNCA may hold great promise for disease intervention in PD.
PMCID: PMC3748195  PMID: 23715007
alpha-synuclein; Parkinson disease; TFEB; MIR128; temsirolimus; dopamine; macroautophagy; neurodegeneration
40.  Functions of autophagy in normal and diseased liver 
Autophagy  2013;9(8):1131-1158.
Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.
PMCID: PMC3748187  PMID: 23774882
autophagy; liver; hepatocyte; hepatitis; ischemia/reperfusion; liver injury; hepatotoxin; hepatocellular carcinoma; drug toxicity
41.  The intense gravitational attraction of autophagy 
Autophagy  2013;9(8):1127-1128.
In the course of my work as Autophagy editor, I try to gauge the overall patterns of interest in autophagy research. Not surprisingly, the number of papers associated with this topic has increased steadily. However, that trend provides only one glimpse into the way interest in this field has been changing—that the number of people working on autophagy has expanded. Perhaps not surprisingly, the number of different research areas that now include autophagy studies is also increasing. Thus, I decided to carry out an informal, imprecise analysis of the number of different journals (presumably reflecting in part the number of topics) that include papers on autophagy.
PMCID: PMC3748185
autophagy; black hole; gravity; lysosome; stress; vacuole
42.  The CFATG (Club Francophone de l’Autophagie, or The Francophone Autophagy Club) 
Autophagy  2013;9(8):1129-1130.
PMCID: PMC3748186  PMID: 23787944
43.  Amorphous areas in the cytoplasm of Dendrobium tepal cells 
Autophagy  2013;9(8):1159-1166.
In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy.
PMCID: PMC3748188  PMID: 23823702
amorphous area; autophagosome; autolysosome; cytoplasm; engulfment; membrane; mesophyll; plant; plastid; tepal; vacuole
44.  Autophagy 
Autophagy  2013;9(8):1253-1255.
Autophagy describes the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. Autophagy is essentially required to prevent accumulation of cellular damage and to ensure cellular homeostasis. Indeed, impaired autophagy has been implicated in a variety of different diseases. We examined the role of autophagy in inflammatory bone loss. We demonstrated that autophagy is activated by the pro-inflammatory cytokine tumor necrosis factor (TNF/TNFα) in osteoclasts of patients with rheumatoid arthritis (RA). Autophagy induces osteoclast differentiation and stimulates osteoclast-mediated bone resorption in vitro and in vivo, thereby highlighting autophagy as a novel mediator of TNF-induced bone resorption.
PMCID: PMC3748199  PMID: 23811580
TNFα; arthritis; autophagy; bone resorption; osteoclasts
45.  Autophagy receptor CALCOCO2/NDP52 takes center stage in Crohn disease 
Autophagy  2013;9(8):1256-1257.
To advance understanding of the complex genetics of Crohn disease (CD) we sequenced 42 whole exomes of patients with CD and five healthy control individuals, resulting in identification of a missense mutation in the autophagy receptor calcium binding and coiled-coil domain 2 (CALCOCO2/NDP52) gene. Protein domain modeling and functional studies highlight the potential role of this mutation in controlling NFKB signaling downstream of toll-like receptor (TLR) pathways. We summarize our recent findings and discuss the role of autophagy as a major modulator of proinflammatory signaling in the context of chronic inflammation.
PMCID: PMC3748200  PMID: 23820297
Crohn disease; autophagy; CALCOCO2; NDP52; inflammation; NF-kappaB; toll-like receptor; adaptophagy
46.  Reciprocal effects of rab7 deletion in activated and neglected T cells 
Autophagy  2013;9(7):1009-1023.
Mouse models lacking proteins essential for autophagosome formation have demonstrated that autophagy plays a critical role in T cell development and activation. To better understand the function of autophagy in quiescent and activated lymphocytes, we have generated a mouse deficient in rab7 selectively in T cells and compared the effects of blocking autophagy at an early (atg5−/−) or late (rab7−/−) stage on T cell biology. rab7−/− murine embryonic fibroblasts (MEFs) and T cells generated from these mice exhibit a profound block in autophagosome degradation and are as sensitive as atg5−/− cells to extracellular nutrient limitation. Rab7flox/floxCD4-Cre+ mice lacking the RAB7 protein in both CD4 and CD8 T cells had reduced numbers of peripheral T cells, but this defect was not as severe as in Atg5flox/floxCD4-Cre+ mice despite efficient rab7 deletion and inhibition of autophagic flux. This difference may stem from the reduced ROS generation and enhanced survival of rab7−/− T cells compared with wild-type and atg5−/− T cells in the absence of cytokine stimulation. rab7−/− and atg5−/− T cells exhibited similar defects in proliferation both following antibody-mediated T cell receptor (TCR) cross-linking and using a more physiologic activation protocol, allogeneic stimulation. Interestingly, autophagy was not required to provide building blocks for the upregulation of nutrient transporter proteins immediately following activation. Together, these studies suggest that autophagosome degradation is required for the survival of activated T cells, but that loss of rab7 is better tolerated in naïve T cells than the loss of atg5.
PMCID: PMC3722312  PMID: 23615463
RAB7; T cells; autophagy; lysosomal fusion; growth factor withdrawal; ATG5
47.  Live-cell imaging of Aspergillus nidulans autophagy 
Autophagy  2013;9(7):1024-1043.
We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This ‘autophagosome cycle’ gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabSRAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabSRAB7 or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabARAB5/RabBRAB5 (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabORAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabORAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabORAB1 are dissociable by mutation: rabOA136D hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.
PMCID: PMC3722313  PMID: 23722157
autophagosome; phagophore; intracellular traffic; nitrogen starvation; Rab; SNARE; Atg8
48.  Lumenal peroxisomal protein aggregates are removed by concerted fission and autophagy events 
Autophagy  2013;9(7):1044-1056.
We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11.
PMCID: PMC3722314  PMID: 23614977
yeast; peroxisome; protein aggregate; fission; autophagy
49.  Harnessing autophagy for cell fate control gene therapy 
Autophagy  2013;9(7):1069-1079.
We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.
PMCID: PMC3722316  PMID: 23633667
autophagy; DTYMK/TMPK; rapamycin; cell-fate control; suicide gene
50.  Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity 
Autophagy  2013;9(7):1080-1093.
Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.
PMCID: PMC3722317  PMID: 23697944
autophagy; polyamines; Trypanosoma cruzi; spermidine; DFMO; autophagic response; LC3; ATG5

Results 26-50 (694)