Showing papers on "Autolysosome published in 2011"
TL;DR: Current evidence strongly points to disruption of substrate proteolysis within autolysosomes for the principal mechanism underlying autophagy failure in AD, and attempts to restore more normal lysosomal proteolytic efficiency in mouse models of AD pathology have yielded promising therapeutic effects.
554 citations
TL;DR: Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP, however, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed.
Abstract: Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors,...
310 citations
TL;DR: It is reported that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells, and this results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection.
Abstract: Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-β activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.
306 citations
TL;DR: The impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s is summarized.
Abstract: Autophagy was first discovered by transmission electron microscopy more than 50 years ago. For decades, electron microscopy was the only way to reliably detect autophagic compartments in cells because no specific protein markers were known. In the 1970s, however, the introduction of biochemical methods enabled quantitative studies of autophagic-lysosomal degradation, and in the 1980s specific biochemical assays for autophagic sequestration became available. Since the identification of autophagy-related genes in the 1990s, combined fluorescence microscopy, biochemical and genetic methods have taken the leading role in autophagy research. However, electron microscopy is still needed to confirm and verify results obtained by other methods, and also to produce novel knowledge that would not be achievable by any other experimental approach. Confocal microscopy, with its ever-improving resolution, is probably the best-suited morphological approach to investigate the dynamic aspects of autophagy. However, for analyzing the ultrastructural details of the many novel organelles and mechanisms involved in specific subtypes of autophagy, the electron microscope is still indispensable. This review will summarize the impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s. Astonishingly, some of the "novel" concepts and principles of autophagy, presented in the recent studies, were already proposed several decades ago by the pioneering, accurate and passionate work of virtuoso electron microscopists.
241 citations
TL;DR: It is found that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation and it is demonstrated that the sugar transporter activity of Spin isessential for ALR.
Abstract: Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the “target of rapamycin” (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. Here we show that defects in spin lead to the accumulation of enlarged autolysosomes. We find that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, we demonstrate that the sugar transporter activity of Spin is essential for ALR.
238 citations
TL;DR: Investigation of lumbar spinal cords in sporadic ALS found that autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegenerating motor neurons in sporadicALS.
Abstract: To assess the potential role of autophagy in amyotrophic lateral sclerosis (ALS), lumbar spinal cords in a total of 19 sporadic ALS cases and 27 age-matched controls were investigated. Immunohistochemical analysis using antibodies to the markers of autophagy microtubule-associated protein light chain 3 (LC3) and p62 was performed on samples from 12 ALS and 15 controls. Electron microscopy was performed on samples from 16 ALS and 15 controls, including overlapping cases. In the ALS cases, the somata of normal-appearing and degenerated motor neurons and round bodies were occasionally immunostained for LC3; round bodies and skein-like inclusions were immunostained for p62. By electron microscopy, all 16 ALS patients showed features of autophagy in the cytoplasm of normal-appearing motor neurons and, more frequently, in degenerated motor neurons. Autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane contained sequestered cytoplasmic organelles, such as mitochondria and ribosome-like structures. These autophagy features were also found in close association with the characteristic inclusions of ALS(i.e. round bodies, skein-like inclusions, and Bunina bodies); honeycomb-like structures also occasionally showed autophagy-associated features. Normal-appearing anterior horn neurons in control patients showed no autophagy features. Thus, autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegeneration of motor neurons in sporadic ALS.
224 citations
TL;DR: It is suggested that HCV harnesses autophagy to circumvent the harmful vacuole formation and to maintain a persistent infection, and new insights are provided into the genotype-specific pathogenicity of HCV.
Abstract: Hepatitis C virus (HCV) is a major cause of chronic liver diseases. A high risk of chronicity is the major concern of HCV infection, since chronic HCV infection often leads to liver cirrhosis and hepatocellular carcinoma. Infection with the HCV genotype 1 in particular is considered a clinical risk factor for the development of hepatocellular carcinoma, although the molecular mechanisms of the pathogenesis are largely unknown. Autophagy is involved in the degradation of cellular organelles and the elimination of invasive microorganisms. In addition, disruption of autophagy often leads to several protein deposition diseases. Although recent reports suggest that HCV exploits the autophagy pathway for viral propagation, the biological significance of the autophagy to the life cycle of HCV is still uncertain. Here, we show that replication of HCV RNA induces autophagy to inhibit cell death. Cells harboring an HCV replicon RNA of genotype 1b strain Con1 but not of genotype 2a strain JFH1 exhibited an incomplete acidification of the autolysosome due to a lysosomal defect, leading to the enhanced secretion of immature cathepsin B. The suppression of autophagy in the Con1 HCV replicon cells induced severe cytoplasmic vacuolation and cell death. These results suggest that HCV harnesses autophagy to circumvent the harmful vacuole formation and to maintain a persistent infection. These findings reveal a unique survival strategy of HCV and provide new insights into the genotype-specific pathogenicity of HCV.
70 citations
TL;DR: The results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.
Abstract: The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.
55 citations
TL;DR: In this review, what is currently known about the interplay between autophagy and HCV is summarized and the possible mechanisms whereby autophile proteins might favor HCV propagation are summarized.
Abstract: Autophagy is a cellular process that catabolizes cytoplasmic components and maintains energy homeostasis. As a stress response, the autophagy machinery interconnects a wide range of cellular pathways, enhancing the spread of certain pathogens while limiting others, and has become a highly active research area over the past several years. Independent laboratories have recently reported that autophagy vesicles accumulate in hepatitis C virus (HCV) infected cells and that autophagy proteins can function as proviral factors required for HCV replication. In this review, we summarize what is currently known about the interplay between autophagy and HCV and the possible mechanisms whereby autophagy proteins might favor HCV propagation.
53 citations
TL;DR: It is concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy, despite being involved in cell growth and autophagy through mammalian target of rapamycin complex 1.
50 citations
TL;DR: Recent studies reveal that virus-induced UPR may activate autophagy to support the virus life cycle, and the exact roles of the UPR and autophagosome in host cell-virus interactions are still enigmatic.
Abstract: Autophagy is an evolutionarily conserved process that catabolizes intracellular components and maintains cellular homeostasis. Autophagy involves the sequestration of cytoplasmic content within a double-membraned autophagosome, and the fusion of the autophagosome with a lysosome to form an autolysosome for subsequent degradation (Fig. 1A). Autophagy plays a pivotal role in various aspects of cellular responses to stresses, such as nutrient deprivation, damaged organelles, aggregated proteins, exposure to endoplasmic reticulum (ER) stress and pathogen infections. Virus infection often leads to ER stress and induction of the unfolded protein response (UPR). Recent studies reveal that virus-induced UPR may activate autophagy to support the virus life cycle. However, the exact roles of the UPR and autophagy in host cell-virus interactions are still enigmatic.
TL;DR: IFN beta mediates a distinct autophagic mechanism of antiviral host defense, which has an important role in type I IFN-induced autophotic trafficking of viral proteins.
TL;DR: The results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes, and indicates that a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.
Abstract: Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.
TL;DR: It is reported that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells, and this results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection.
TL;DR: The study demonstrated that monomeric red fluorescence protein-light chain 3 (mRFP-LC3)-labeled autolysosomes were motile and moved along PC12 neurites in both anterograde and retrograde directions with a bias towards the nucleus during starvation.
TL;DR: The hypothesis that plastids in Dendrobium petal mesophyll cells can function in a way similar to both autophagosomes and autolysosomes in animal cells is suggested.
Abstract: In animal cells a double-membrane-bound structure, the autophagosome, encloses a portion of the cytoplasm. The encapsulated material becomes digested after fusion of the autophagosome with a vesicle containing lytic enzymes. The autophagosome is then termed autolysosome. In intact plants, structures similar to animal autophagosomes/autolysosomes have been found only in a few types of cells. Additionally, some early papers indicated that plastids can function similar to autophagosomes/autolysosomes. Here, we report that plastids in Dendrobium cv. Lucky Duan petals produced an endocytosis-like invagination of the two outer membranes. The opening between the invagination space and the cytoplasm was almost isodiametric, less than 0.2 μm in diameter. The volume of the space formed by the invagination had a maximum of about half of the total plastid volume. Staining of the invagination lumen for acid phosphatase, a marker of organelles showing autophagic activity, was positive. Membranes and numerous ribosomes were observed inside the lumen of the invagination. The structure of the material inside the lumen varied from that of the cytoplasm to uniform electron-translucent, indicating that the enclosed cytoplasmic material became completely digested. No support was found for the idea that the material engulfed by the plastid or the whole plastid became transferred to a vacuole. Taken together, the data suggested the hypothesis that plastids in Dendrobium petal mesophyll cells can function in a way similar to both autophagosomes and autolysosomes in animal cells.
Journal Article•
TL;DR: Iradiation may induce the autophagy in human bone marrow mesenchymal stem cells (hBMMSC), and autophagic vacuoles could protect h BMMSC from irradiation injury in a certain dose range.
Abstract: OBJECTIVE To investigate the autophagy in human bone marrow mesenchymal stem cells (hBMMSC) exposed to irradiation. METHODS The apoptosis and necrosis rate were assessed by Annexin V and propidium (PI) staining in hBMMSC at 4h after irradiated with X-ray at 0, 2, 4, 8 and 10 Gy. The autophagy was observed by transmission electron microscopy. The mRNA expression of Beclin1 and microtubule-associated protein 1 light chain 3 (MAPLC3 or LC3) was analyzed by RT-PCR in hBMMSC at 4h after X-ray irradiation at 0, 8 and 10 Gy. RESULTS The apoptosis rate of hBMMSC was markedly decreased while the necrosis and death rate were slowly increased with the increase of irradiation dose when under 8 Gy. The apoptosis rate was significantly increased and reached a peak while the necrosis and whole death rate were obviously increased when irradiated with 10 Gy X-rays. In addition, the change of apoptosis rate was more significant than that of necrosis rate. By electron microscopy, a mass of autophagic vacuoles (autophagosome and autolysosome) were observed in irradiation and positive control groups, but were only occasionally seen in normal control group. The proportion of hBMMSC with autophagic vacuoles in 8 Gy irradiation group was higher than that in 10 Gy one. The mRNA expression of Beclin1 and LC3 in irradiation groups and positive control group was significantly higher than in normal control group, and so did in 8 Gy irradiation group than that in 10 Gy group. CONCLUSION Irradiation may induce the autophagy in hBMMSC, and autophagy could protect hBMMSC from irradiation injury in a certain dose range.
TL;DR: An integrated in vitro and in vivo system is established to investigate the biological functions of autophagy in maintaining oxidative metabolism in active Ras-mediated tumorigenesis and the intriguing work reported by Guo et al. impacts on the interplay between Autophagy/mitophagy and mitochondrially oxidative metabolism.
Abstract: Macroautophagy (hereafter referred to as autophagy) or ‘self-eating' is a lysosomal degradation pathway and plays a role in the breakdown of disordered intracellular organelles, such as peroxisomes (pexophagy), mitochondria (mitophagy), endoplasmic reticula (reticulophagy) and ribosomes (ribophagy), as well as providing for controlled recycling of macromolecules during cellular adaption and pathogenesis.1, 2 Mitophagy serves to remove mitochondria containing damaged components and also to eliminate subsets of mitochondria producing reactive oxygen species.3 The lysosomal compartment is responsible for the controlled recycling of cellular organelles and macromolecules. These dynamic organelles contain hydrolytic enzymes (proteases, lipases and glycosidases) capable of degrading proteins, proteoglycans, nucleic acids and lipids. Both heterophagic and autophagic cargos find their final destiny in lysosomes, where they are broken down by numerous hydrolyses.4 Certain environmental cues (such as starvation, high temperature, low oxygen and hormonal stimulation) or intracellular stress (damaged organelles, accumulation of mutant proteins and microbial invasion) activate signaling pathways that increase autophagy.1, 2, 5 When the cell receives an appropriate signal, autophagy-execution proteins trigger a cascade of reactions that result in the formation of double membrane-bound vesicles called autophagosomes. The vesicles then fuse with lysosomes followed by a release of lysosomal digestive enzymes into the lumen of the resulting autolysosomes. The sequestered cytoplasmic contents are degraded inside the autolysosome into free nucleotides, amino acids and fatty acids, which are reused by the cell to maintain macromolecular synthesis and to fuel energy production.6 Autophagy is induced in vivo in tumors in hypoxic regions and contributes to tumor cell survival.7 Accumulated defective lysosomes and autophagic vacuoles were detected in both nuclear receptor PPARγ- and PPARγ2-deficient prostatic carcinogenesis.8, 9 Autophagy is also frequently activated in different tumor cells treated with chemotherapy or irradiation. Short-term inhibition of autophagy along with radiotherapy leads to enhanced cytotoxicity of radiotherapy in resistant cancer cells. Autophagy acts either to destroy defective cells, or as a survival mechanism for damaged cells putting them in a position to accumulate further genetic damage, suggestive of ‘a double-edged of sword' reported in different types of cancer.10 Whether autophagy is ‘protective' for the organism by promoting effective ‘self-eating and self-digesting' and/or ‘self-killing' of damaged cells or alternatively, acts as an ‘oncogenic' survival response in cancer is not yet determined. Recently in an original research paper published in Genes & Development, Guo et al. hypothesized that autophagy plays opposing roles in tumor initiation and in established human tumors.11 They suggested that, whereas damage mitigation resulting from autophagy may be important for suppressing tumor initiation, in aggressive cancers, growth in a stressed microenvironment may instead result in dependency on autophagy for survival. The intriguing work reported by Guo et al. impacts on the interplay between autophagy/mitophagy and mitochondrially oxidative metabolism in a model of Ras mutations (H-rasV12 or K-rasV12)-induced tumorigenesis. The authors have established an integrated in vitro and in vivo system to investigate the biological functions of autophagy in maintaining oxidative metabolism in active Ras-mediated tumorigenesis.