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issn:1554-86.7
1.  Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a two-dimensional matched filter 
Autophagy  2013;10(1):10.4161/auto.26678.
Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive and not always completely reliable. To enable label-free analyses we introduce a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein and analysis of the autophagic flux. Selected detection results for two additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
doi:10.4161/auto.26678
PMCID: PMC3872480  PMID: 24169509
phase-contrast microscopy; automated microscopy; vesicle detection; autophagy; image processing
2.  Xenophagy in herpes simplex virus replication and pathogenesis 
Autophagy  2007;4(1):101-103.
Autophagy functions in part as an important host defense mechanism to engulf and degrade intracellular pathogens, a process that has been termed xenophagy. Xenophagy is detrimental to the invading microbe in terms of replication and pathogenesis and many pathogens either dampen the autophagic response, or utilize the pathway to enhance their life cycle. Herpes simplex virus type 1 (HSV-1) counteracts the induction of xenophagy through its neurovirulence protein, ICP34.5. ICP34.5 binds protein phosphatase 1a to counter PKR-mediated phosphorylation of eIF2α, and also binds the autophagy-promoting protein Beclin 1. Through these interactions, ICP34.5 prevents translational arrest and downregulates the formation of autophagosomes. Whereas autophagy antagonism promotes neurovirulence, it has no impact on the replication of HSV-1 in permissive cultured cells. As discussed in this article, this work raises a number of questions as to the mechanism of ICP34.5-mediated inhibition of autophagy, as well as to the role of autophagy antagonism in the lifecycle of HSV-1.
PMCID: PMC3775468  PMID: 18000391
autophagy; innate immunity; herpes simplex virus; pathogenesis; immunomodulation
3.  Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells 
Autophagy  2010;6(4):462-472.
Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) then cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.
doi:10.4161/auto.6.4.11553
PMCID: PMC3768271  PMID: 20364102
autophagy; cardiac myocyte; cyclophilin D; mitochondrial permeability transition
4.  Quality control autophagy—a joint effort of ubiquitin, protein deacetylase and actin cytoskeleton 
Autophagy  2010;6(4):555-557.
Summary
Autophagy has been predominantly studied as a non-selective self-digestion process that recycles macromolecules and produces energy in response to starvation. However, autophagy independent of nutrient status has long been known to exist. Recent evidence suggests that this form of autophagy enforces intracellular quality control by selectively disposing of aberrant protein aggregates and damaged organelles – common denominators in various forms of neurodegenerative diseases. By definition, this form of autophagy, termed quality-control (QC) autophagy, must be different from nutrient-regulated autophagy in substrate selectivity, regulation, and function. We have recently identified the ubiquitin-binding deacetylase, HDAC6, as a key component that establishes QC. HDAC6 is not required for autophagy activation per se; rather, it is recruited to ubiquitinated autophagic substrates where it stimulates autophagosome-lysosome fusion by promoting F-actin remodeling in a cortactin-dependent manner. Remarkably, HDAC6 and cortactin are dispensable for starvation-induced autophagy. These findings reveal that autophagosomes associated with QC are molecularly and biochemically distinct from those associated with starvation autophagy, thereby providing a new molecular framework to understand the emerging complexity of autophagy and therapeutic potential of this unique machinery.
doi:10.4161/auto.6.4.11812
PMCID: PMC3752377  PMID: 20404488
5.  Eat your heart out 
Autophagy  2008;4(4):416-421.
Autophagy is an important process in the heart which is responsible for the normal turnover of long lived proteins and organelles. Inhibition of autophagy leads to the accumulation of protein aggregates and dysfunctional organelles which can cause cell death. Autophagy occurs at low basal levels under normal conditions in the heart, but is rapidly upregulated in response to stress such as nutrient deprivation, hypoxia and pressure overload. Autophagy is a prominent feature of myocardial ischemia and reperfusion. Although enhanced autophagy is often seen in dying cardiac myocytes, the functional significance of autophagy under these conditions is not clear. Upregulation of autophagy has been reported to protect cardiac cells against death as well as be the cause of it. Here, we review the evidence that autophagy can have both beneficial and detrimental roles in the myocardium, and discuss potential mechanisms by which autophagy provides protection in cells.
PMCID: PMC3723414  PMID: 18253087
autophagy; ischemia; reperfusion; heart; cardiac myocytes; mitochondria; cell death; beclin 1
6.  A Method to Measure Cardiac Autophagic Flux in vivo 
Autophagy  2008;4(3):322-329.
Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 μM and 8 μM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.
PMCID: PMC3709927  PMID: 18216495
chloroquine; autophagy; cardiac myocytes; LC3; monodansylcadaverine
7.  Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD 
Autophagy  2010;6(4):523-541.
Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFNα permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin 1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.
doi:10.4161/auto.6.4.11811
PMCID: PMC3707503  PMID: 20404486
Th1/Tc1; rapamycin; autophagy; apoptosis resistance; xenogeneic GVHD
8.  Using LC3 to Monitor Autophagy Flux in the Retinal Pigment Epithelium 
Autophagy  2009;5(8):1190-1193.
Summary
Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation.1,2 Autophagy begins with the formation of membranes arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. 1,2,7 However, few studies of autophagy in retinal disease have been reported.
Recent studies show that autophagy and changes in lysosomal activity are associated with both retinal aging and age-related macular degeneration (AMD).3,4 This article describes methods which employ the target protein LC3 to monitor autophagic flux in retinal pigment epithelial cells. During autophagy, the cytosolic form of LC3 (LC3-I) is processed and recruited to the phagophore where it undergoes site specific proteolysis and lipidation near the C terminus to form LC3-II.5 Monitoring the formation of cellular autophagosome puncta containing LC3 and measuring the ratio of LC3-II to LC3-I provides the ability to monitor autophagy flux in the retina.
PMCID: PMC3704326  PMID: 19855195
autophagy flux; LC3; retinal pigment epithelium; lysosomes; age-related macular degeneration; aging
9.  Autophagic control of RLR signaling 
Autophagy  2009;5(5):749-750.
Innate immunity to viral infection is initiated within the infected cells through the recognition of unique viral signatures by pattern recognition receptors (PRRs) that mediate the induction of potent antiviral factor, type I interferons (IFNs). Infection with RNA viruses is recognized by the members of the retinoic acid inducible gene I (RIG-I)-like receptor (RLR) family in the cytosol. Our recent study demonstrates that IFN production in response to RNA viral ligands is increased in the absence of autophagy. The process of autophagy functions as an internal clean-up crew within the cell, shuttling damaged cellular organelles and long-lived proteins to the lysosomes for degradation. Our data show that the absence of autophagy leads to the amplification of RLR signaling in two ways. First, in the absence of autophagy, mitochondria accumulate within the cell leading to the build up of mitochondrial associated protein, IPS-1, a key signaling protein for RLRs. Second, damaged mitochondria that are not degraded in the absence of autophagy provide a source of reactive oxygen species (ROS), which amplify RLR signaling in Atg5 knockout cells. Our study provides the first link between ROS and cytosolic signaling mediated by the RLRs, and suggests the importance of autophagy in the regulation of signaling emanating from mitochondria.
PMCID: PMC3693554  PMID: 19571662
10.  Autophagy protects from hypoxic injury in C. elegans 
Autophagy  2008;4(8):1034-1041.
Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after an hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell death mechanisms is inhibited.
PMCID: PMC3670992  PMID: 18849662
Autophagy; Cell death; Hypoxia; Apoptosis; Necrosis
11.  Artophagy 
Autophagy  2010;6(1):3-6.
Science informs art, and art informs science. Both processes involve creativity and imagination, and collaboration between scientists and artists often leads to new insights in both fields. We took advantage of the power of artistic imagery to demonstrate a dynamic cellular process, autophagy. In particular, we depicted the cytoplasm to vacuole targeting pathway, which involves dynamic membrane rearrangements to sequester a specific cargo via an autophagy-related process. By depicting this event in the context of a crowded cellular milieu, we hoped to stimulate researchers to consider aspects of the process that might be overlooked in the overly simplistic schematic drawing that typify most scientific models.
PMCID: PMC3655401  PMID: 20023390
collaboration; Cvt complex; membrane; molecular model; organelle; science
12.  Autophagy receptors in developmental clearance of mitochondria 
Autophagy  2011;7(3):301-303.
Recent discoveries of autophagy receptors, which specifically recognize different cellular cargo destined for degradation, have opened a new chapter in the autophagy field. Selective cargo recognition by autophagic machinery is important in the context of cellular homeostasis and survival. One of the crucial homeostasis events involving autophagy is the removal of damaged or excessive mitochondria through mitophagy. Future studies on mitochondrial receptors and proteins associated with mitochondrial clearance will help us better understand the role of mitophagy in normal physiological processes as well as in diverse pathological conditions.
PMCID: PMC3654195  PMID: 21206218
mitophagy; autophagy receptors; mitochondria; Nix; Bnip3; reticulocyte maturation
13.  Analyzing autophagy in zebrafish 
Autophagy  2010;6(5):642-644.
The transparency, external development and simple drug administration of zebrafish embryos makes them a useful model for studying autophagy during embryonic development in vivo. Cloning of zebrafish lc3 and generation of a transgenic GFP-Lc3 fish line provide excellent tools to monitor autophagy in this organism.1 This protocol discusses several convenient autophagy assays in zebrafish, including immunoblotting of Lc3 lipidation, microscopy imaging of GFP-Lc3 and lysosomal staining.
doi:10.4161/auto.6.5.12092
PMCID: PMC3654832  PMID: 20495344
lysosome; protein degradation; protein targeting; stress; vacuole
14.  A comprehensive glossary of autophagy-related molecules and processes 
Autophagy  2010;6(4):438-448.
Autophagy is a rapidly expanding field in the sense that our knowledge about the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. Similarly, the vocabulary associated with autophagy has grown concomitantly. This fact makes it difficult for readers, even those who work in the field, to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors or chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, or the role of accessory machinery or structures that are associated with autophagy.
doi:10.4161/auto.6.4.12244
PMCID: PMC3652604  PMID: 20484971
autophagy; definitions; glossary; lexicon; terms
15.  Autophagy is required for extension of yeast chronological life span by rapamycin 
Autophagy  2009;5(6):847-849.
Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.
PMCID: PMC3586265  PMID: 19458476
autophagy; aging; chronological life span; rapamycin; Saccharomyces cerevisiae
16.  E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells 
Autophagy  2011;7(12):1566-1567.
The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.
doi:10.4161/auto.7.12.17991
PMCID: PMC3288034  PMID: 22024746
E4F1; histiocytic sarcoma; mitochondria; autophagy; cell death; leukemic cells; reactive oxygen species
17.  Viral evasion of autophagy 
Autophagy  2007;4(3):280-285.
Autophagy is an evolutionarily ancient pathway for survival during different forms of cellular stress, including infection with viruses and other intracellular pathogens. Autophagy may protect against viral infection through degradation of viral components (xenophagy), by promoting the survival or death of infected cells, through delivery of Toll-like receptor (TLR) ligands to endosomes to activate innate immunity, or by feeding antigens to MHC class II compartments to activate adaptive immunity. Given this integral role of autophagy in innate and adaptive antiviral immunity, selective pressure likely promoted the emergence of escape mechanisms by pathogenic viruses. This review will briefly summarize the current understanding of autophagy as an antiviral pathway, and then discuss strategies that viruses may utilize to evade this host defense mechanism.
PMCID: PMC3508671  PMID: 18059171
autophagy; virus; xenophagy; immunity; viral evasion
18.  Ceramide-induced autophagy 
Autophagy  2009;5(4):558-560.
Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased. It is notable that the conversion of short-chain ceramides (C2-ceramide and C6-ceramide) into long-chain ceramide via the activity of ceramide synthase is required to trigger autophagy. The dissociation of the Beclin 1:Bcl-2 complex has also been observed in response to tamoxifen and PDMP (an inhibitor of the enzyme that converts ceramide to glucosylceramide), drugs that increase the intracellular level of long-chain ceramides. However, and in contrast to starvation, over-expression of Bcl-2 does not blunt ceramide-induced autophagy. Whether this autophagy that is unchecked by forced dissociation of the Beclin 1:Bcl-2 complex is related to the ability of ceramide to trigger cell death remains an open question. More generally, the question of whether ceramide-induced autophagy is a dedicated cell death mechanism deserves closer scrutiny.
PMCID: PMC3501009  PMID: 19337026
macroautophagy; Bcl-2; Beclin 1; c-Jun N-terminal kinase; cell death; sphingolipids
19.  E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells 
Autophagy  2011;7(12):1566-1567.
Summary
The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.
PMCID: PMC3288034  PMID: 22024746
Animals; Autophagy; Cell Survival; Cell Transformation, Neoplastic; pathology; Disease Models, Animal; Humans; Leukemia, Myeloid; metabolism; pathology; Mice; RNA, Small Interfering; metabolism; Reactive Oxygen Species; metabolism; Repressor Proteins; metabolism; E4F1; histiocytic sarcoma; mitochondria; autophagy; cell death; leukemic cells; reactive oxygen species
20.  Autophagy discriminates between Alix and ESCRTs 
Autophagy  2009;5(1):106-107.
SUMMARY
Alix and ESCRT proteins are required for membrane fission during viral budding and egress and during the abscission stage of cytokinesis. These common roles have suggested that Alix functions as an ESCRT protein, a conclusion challenged by the finding that unlike ESCRTs, which control the formation of multivesicular endosomes, Alix does not influence the degradation of the EGF receptor. We previously showed that Alix controls neuronal death by an unknown mechanism, but dependent on its interaction with ESCRT proteins. Since then, numerous reports have shown that ESCRTs participate in macroautophagy. Given the direct interaction between ESCRTs and Alix, together with the known contribution of autophagy to cell death, it was hypothesized that Alix controls autophagy and thereby cell death. Our recent published results show that this is not the case. ESCRT protein activity therefore needs Alix for viral budding and cytokinesis but not for autophagy. The function of ESCRT can thus be clearly be disconnected from that of Alix.
PMCID: PMC3325908  PMID: 19029801
Animals; Autophagy; Biological Transport; Calcium-Binding Proteins; metabolism; Caspases; metabolism; Endosomes; metabolism; Humans; Multiprotein Complexes; metabolism; Alix; Autophagy; ESCRT; endocytosis; MVB
21.  The Atg6/Vps30/Beclin1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans 
Autophagy  2011;7(4):386-400.
Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identified a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. similarly, we found that lack of bec-1 also results in mislocalization and degradation of MIG-14∷GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identified a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we found that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.
PMCID: PMC3108013  PMID: 21183797
C. elegans; autophagy; endocytosis; lysosomes
22.  The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans 
Autophagy  2011;7(4):386-400.
Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin 1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identified a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we found that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identified a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we found that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.
doi:10.4161/auto.7.4.14391
PMCID: PMC3108013  PMID: 21183797
C. elegans; autophagy; endocytosis; lysosomes
23.  Regulation of autophagy by ceramide-CD95-PERK signaling 
Autophagy  2008;4(7):929-931.
The manuscripts by Park et al.1 and Zhang et al.2 were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor–induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2α-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
PMCID: PMC3292039  PMID: 18719356
Vorinostat; Sorafenib; bile acid; CD95; autophagy; ceramide; cell death; ASMase
24.  ATM engages the TSC2/mTORC1 signaling node to regulate autophagy 
Autophagy  2010;6(5):672-673.
doi:10.4161/auto.6.5.12509
PMCID: PMC3259740  PMID: 20581436
ATM; cytoplasm; ROS; mTOR; DNA damage
25.  When more is less 
Autophagy  2009;5(1):111-113.
The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, which results from a deficiency of the glycogen-degrading lysosomal hydrolase acid-alpha glucosidase (GAA). Skeletal and cardiac muscle are most severely affected by the progressive expansion of glycogen-filled lysosomes. In both humans and an animal model of the disease (GAA KO), skeletal muscle pathology also involves massive accumulation of autophagic vesicles and autophagic buildup in the core of myofibers, suggesting an induction of autophagy. Only when we suppressed autophagy in the skeletal muscle of the GAA KO mice did we realize that the excess of autophagy manifests as a functional deficiency. This failure of productive autophagy is responsible for the accumulation of potentially toxic aggregate-prone ubiquitinated proteins, which likely cause profound muscle damage in Pompe mice. Also, by generating muscle-specific autophagy-deficient wild-type mice, we were able to analyze the role of autophagy in healthy skeletal muscle.
PMCID: PMC3257549  PMID: 19001870
Pompe disease; lysosome; muscle-specific autophagy deficiency; protein inclusions

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