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1.  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
2.  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
3.  PINK1-parkin-dependent mitophagy involves ubiquitination of mitofusins 1 and 2 
Autophagy  2011;7(2):243-245.
PMCID: PMC4196638  PMID: 21139416
mitochondria; mitofusin; mitophagy; neurodegeneration; Parkinson disease; parkin; PINK1
4.  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
5.  Activation of autophagy is required for muscle homeostasis during physical exercise 
Autophagy  2011;7(12):1405-1406.
Skeletal muscle fibers of collagen VI null (Col6a1−/−) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.
doi:10.4161/auto.7.12.18315
PMCID: PMC3288013  PMID: 22082869
lysosome; metabolism; physiology; stress; vacuole
6.  Highlights from this issue 
Autophagy  2011;7(12):1407-1409.
doi:10.4161/auto.7.12.18505
PMCID: PMC3288014
7.  A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy 
Autophagy  2011;7(12):1410-1414.
A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.1 Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,2,3 and the amount of Atg8 in part determines the size of the autophagosome,4 whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.5 Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.
doi:10.4161/auto.7.12.16991
PMCID: PMC3288015  PMID: 22024756
autophagy; collaboration; gel electrophoresis; membrane; primer
8.  Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles 
Autophagy  2011;7(12):1415-1423.
Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1–/–) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1–/– mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1–/– muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity.
doi:10.4161/auto.7.12.17877
PMCID: PMC3288016  PMID: 22024752
autophagy; muscle; muscular dystrophy; mouse model; collagen VI
9.  Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation 
Autophagy  2011;7(12):1448-1461.
Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.
doi:10.4161/auto.7.12.17802
PMCID: PMC3288019  PMID: 22082875
WIPI-1; Atg18; PtdIns(3)P; LC3; resveratrol; noncanonical autophagy
10.  Accumulation of p62 in degenerated spinal cord under chronic mechanical compression 
Autophagy  2011;7(12):1462-1471.
Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.
doi:10.4161/auto.7.12.17892
PMCID: PMC3288020  PMID: 22082874
p62; autophagy; cervical spondylotic myelopathy; tiptoe-walking Yoshimura (twy) mice; ubiquitinated proteins
11.  Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex 
Autophagy  2011;7(12):1500-1513.
A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.
doi:10.4161/auto.7.12.18025
PMCID: PMC3288023  PMID: 22082872
autophagy; Atg16L; autophagosome; coiled-coil domain; LC3; Rab33-binding protein; Rab effector
12.  Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis 
Autophagy  2011;7(12):1514-1527.
The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.
doi:10.4161/auto.7.12.18040
PMCID: PMC3288024  PMID: 22082871
atg5; zebrafish; autophagy; neurogenesis; expression regulation; feedback inhibition; neural gene; rapamycin
13.  Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells 
Autophagy  2011;7(12):1528-1545.
Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.
doi:10.4161/auto.7.12.18051
PMCID: PMC3288025  PMID: 22108004
Alzheimer disease; amyloid β-protein; amyloid precursor protein; apoptosis; autophagy; lysosomes; oxidative stress
14.  Evolution of the “autophagamiR” 
Autophagy  2011;7(12):1553-1554.
MicroRNAs (miRs) are increasingly important diagnostic and prognostic markers in cancer but have not been defined in medullary thyroid carcinoma (MTC). MiR microarray profiling was performed on 19 primary MTC tumors, validated with qPCR in 45 cases and correlated with clinical outcomes. MiRs-183 and 375 were overexpressed and miR-9* underexpressed in sporadic vs. hereditary MTC (SMTC; HMTC). MiR-183 and 375 overexpression predicted lateral nodal metastases, residual disease, distant metastases and mortality. MiR-183 knockdown in an MTC cell line (TT cells) reduced cellular proliferation in association with elevated LC3B expression. This is suggestive of increased autophagic flux and potential cell death via autophagy induction. MiRs may subsequently be shown to serve as efficacious therapeutic strategies in MTC with a mechanism based upon autophagy.
doi:10.4161/auto.7.12.17762
PMCID: PMC3288028  PMID: 22024754
autophagamiR; autophagy; miR; microRNA; LC3B
15.  Phospholipid synthetic defect and mitophagy in muscle disease 
Autophagy  2011;7(12):1559-1561.
Mitophagy, selective autophagy of mitochondria, has been extensively demonstrated in cultured cell models but has never been described in skeletal muscle in the context of muscle disease. We recently reported the first example of human muscle disease where mitophagy plays a role in the peculiar muscle pathology. This disease is caused by loss-of-function mutations in the CHKB gene encoding choline kinase β. “Patients” and rostrocaudal muscular dystrophy (rmd) mice, spontaneous Chkb mutants, develop congenital muscular dystrophy with a peculiar mitochondrial abnormality—mitochondria are markedly enlarged at the periphery of muscle fibers and absent from the center. Choline kinase is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. Our discovery demonstrates that a phosphatydilcholine biosynthetic defect leads to mitochondrial dysfunction and increased mitophagy.
doi:10.4161/auto.7.12.17925
PMCID: PMC3288031  PMID: 22024749
choline kinase beta; phosphatidylcholine; mitochondria; mitophagy; congenital muscular dystrophy
16.  Impaired mitophagy at the heart of injury 
Autophagy  2011;7(12):1573-1574.
Recent publications link mitophagy mediated by PINK1 and Parkin with cardioprotection and attenuation of inflammation and cell death. The field is in need of methods to monitor mitochondrial turnover in vivo to support the development of new therapies targeting mitochondrial turnover.
doi:10.4161/auto.7.12.18175
PMCID: PMC3288037  PMID: 22082870
mitophagy; mitochondria; cardiac; ischemia; inflammation; Parkin; cytokine
17.  Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction 
Autophagy  2011;7(12):1424-1433.
Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.
doi:10.4161/auto.7.12.18027
PMCID: PMC3327613
Atg13; autophagy; FIP200; Ulk1; Ulk2
18.  Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation 
Autophagy  2011;7(12):1434-1447.
The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.
doi:10.4161/auto.7.12.17793
PMCID: PMC3327614  PMID: 22024753
ATG12; ATG3; autophagy; LC3 lipidation; vaccinia virus
19.  The induction of autophagy by mechanical stress 
Autophagy  2011;7(12):1490-1499.
The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.
doi:10.4161/auto.7.12.17924
PMCID: PMC3327616  PMID: 22024750
autophagy; homeostasis; mechanical stress; mechanobiology
20.  GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis 
Autophagy  2011;7(12):1546-1550.
Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.
doi:10.4161/auto.7.12.18424
PMCID: PMC3327617  PMID: 22108003
autophagy; lysosome; stress; vacuole; yeast
21.  A subunit of coatomer protein complex offers a novel tumor-specific target through a surprising mechanism 
Autophagy  2011;7(12):1551-1552.
COPI, a coatomer protein complex of secretory vesicles, is involved in Golgi and endoplasmic reticulum traffic and in early endosome maturation. The loss of COPI results in the fragmentation of Golgi, accumulation of immature autophagosomes, inhibition of autophagy, and cell death. Since COPI is required by all cells, it would appear an unlikely target for cancer treatment. However, our recent function-based genomic screen unexpectedly identified a specific COPI subunit, ζ1, as a cancer-specific target. The existing cancer drugs kill only proliferating but not growth-arrested tumor cells, but the depletion of ζ1 induces cell death in both dividing and nondividing tumor cells, while sparing normal cells. The mechanism of this remarkable tumor selectivity turned out to be surprising and heretofore unprecedented.
doi:10.4161/auto.7.12.17659
PMCID: PMC3327618  PMID: 22024755
autophagy; cancer chemotherapy; cancer targets; COPI; COPZ1; COPZ2; function-based genomics; intracellular traffic; microRNA
22.  NAC1 and HMGB1 enter a partnership for manipulating autophagy 
Autophagy  2011;7(12):1557-1558.
Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.
doi:10.4161/auto.7.12.17910
PMCID: PMC3327620  PMID: 22024751
Apoptosis; autophagy; cisplatin; HMGB1; NAC1
23.  Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy 
Autophagy  2011;7(12):1562-1563.
Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of “neuritic dystrophy” in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.
doi:10.4161/auto.7.12.17956
PMCID: PMC3327621  PMID: 22024748
Alzheimer disease; autophagy; axonal transport; dystrophic neurites; lysosomes; proteolysis
24.  MAPKs regulate mitophagy in Saccharomyces cerevisiae 
Autophagy  2011;7(12):1564-1565.
The autophagy-dependent selective degradation of mitochondria (mitophagy) plays an important role in removing excessive, damaged and dysfunctional mitochondria to maintain a proper cellular homeostasis. Relative to its significance in cell physiology, very little is known about the molecular machinery and regulatory mechanism of mitophagy in mammalian cells or yeast. We found that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is involved in both mitophagy and pexophagy (the selective degradation of peroxisomes through autophagy), whereas Hog1 functions specifically in mitophagy.
doi:10.4161/auto.7.12.17971
PMCID: PMC3327622  PMID: 22024747
autophagy; kinase; mitochondria; PAS; regulation; vacuole
25.  Autophagosome biogenesis requires SNAREs 
Autophagy  2011;7(12):1570-1572.
We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.
doi:10.4161/auto.7.12.18001
PMCID: PMC3327624  PMID: 22024744
Atg9; autophagy; lysosome; phagophore assembly site; SNARE; tubulovesicular clusters; vacuole

Results 1-25 (121)