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Autophagy 2018Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic...
UNLABELLED
Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway. Based on the theoretical framework of metabolic control analysis, where the autophagosome flux is the quantitative description of the rate a flow along a pathway, here we treat the autophagy system as a multi-step pathway. We describe a single-cell fluorescence live-cell imaging-based approach that allows the autophagosome flux to be accurately measured. This method characterizes autophagy in terms of its complete autophagosome and autolysosome pool size, the autophagosome flux, J, and the transition time, τ, for autophagosomes and autolysosomes at steady state. This approach provides a sensitive quantitative method to measure autophagosome flux, pool sizes and transition time in cells and tissues of clinical relevance.
ABBREVIATIONS
ATG5/APG5, autophagy-related 5; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; J, flux; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin kinase; nA, number of autophagosomes; nAL, number of autolysosomes; nL, number of lysosomes; p-MTOR, phosphorylated mechanistic target of rapamycin kinase; RFP, red fluorescent protein; siRNA, small interfering RNA; τ, transition time; TEM, transmission electron microscopy.
Topics: Animals; Autophagosomes; Cell Survival; Image Processing, Computer-Assisted; Lysosomes; Mice; Microscopy, Fluorescence; Single-Cell Analysis; Sirolimus; Time Factors
PubMed: 29909716
DOI: 10.1080/15548627.2018.1469590 -
Autophagy Feb 2019Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to...
Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to improve the efficiency of photosensitizers, in terms of inducing cell death, remains unexplored. Here, we addressed the concept that a parallel damage in the membranes of mitochondria and lysosomes leads to a scenario of autophagy malfunction that can greatly improve the efficiency of the photosensitizer to cause cell death. Specific damage to these organelles was induced by irradiation of cells pretreated with 2 phenothiazinium salts, methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB). At a low concentration level (10 nM), only DMMB could induce mitochondrial damage, leading to mitophagy activation, which did not progress to completion because of the parallel damage in lysosome, triggering cell death. MB-induced photodamage was perceived almost instantaneously after irradiation, in response to a massive and nonspecific oxidative stress at a higher concentration range (2 µM). We showed that the parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a late and more efficient cell death, offering significant advantage (2 orders of magnitude) over photosensitizers that cause unspecific oxidative stress. We are confident that this concept can be used to develop better light-activated drugs. ΔΨm: mitochondrial transmembrane inner potential; AAU: autophagy arbitrary units; ATG5, autophagy related 5; ATG7: autophagy related 7; BAF: bafilomycin A; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH: 2',7'-dichlorodihydrofluorescein; DMMB: 1,9-dimethyl methylene blue; ER: endoplasmic reticulum; HaCaT: non-malignant immortal keratinocyte cell line from adult human skin; HP: hydrogen peroxide; LC3B-II: microtubule associated protein 1 light chain 3 beta-II; LMP: lysosomal membrane permeabilization; LTG: LysoTracker™ Green DND-26; LTR: LysoTracker™ Red DND-99; 3-MA: 3-methyladenine; MB: methylene blue; mtDNA: mitochondrial DNA; MitoSOX™: red mitochondrial superoxide probe; MTDR: MitoTracker™ Deep Red FM; MTO: MitoTracker™ Orange CMTMRos; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTT: methylthiazolyldiphenyl-tetrazolium bromide; O: singlet oxygen; OH hydroxil radical; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PBS: phosphate-buffered saline; PI: propidium iodide; PDT: photodynamic therapy; PS: photosensitizer; QPCR: gene-specific quantitative PCR-based; Rh123: rhodamine 123; ROS: reactive oxygen species RTN: rotenone; SQSTM1/p62: sequestosome 1; SUVs: small unilamellar vesicles; TBS: Tris-buffered saline.
Topics: Autophagy; Cell Death; Cell Line; Cell Survival; Humans; Light; Lysosomes; Methylene Blue; Mitochondria; Models, Biological
PubMed: 30176156
DOI: 10.1080/15548627.2018.1515609 -
Autophagy Aug 2021Different types of autophagy co-exist in all mammalian cells, however, the specific contribution of each of these autophagic pathways to the maintenance of cellular... (Review)
Review
Different types of autophagy co-exist in all mammalian cells, however, the specific contribution of each of these autophagic pathways to the maintenance of cellular proteostasis and cellular function remains unknown. In this work, we have investigated the consequences of failure of chaperone-mediated autophagy (CMA) in neurons and compared the impact, on the neuronal proteome, of CMA loss to that of macroautophagy loss. We found that these autophagic pathways are non-redundant and that CMA is the main one responsible for maintenance of the metastable proteome (the one at risk of aggregation). We demonstrate that loss of CMA, as the one that occurs in aging, has a synergistic effect with the proteotoxicity associated with neurodegenerative conditions such as Alzheimer disease (AD) and, conversely, that, pharmacological enhancement of CMA is effective in improving both behavior and pathology in two different AD mouse models.
Topics: Aging; Animals; Autophagy; Chaperone-Mediated Autophagy; Humans; Lysosomes; Neurons; Proteostasis
PubMed: 34110247
DOI: 10.1080/15548627.2021.1935007 -
Autophagy Apr 2019Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune...
Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune system. Increased antibiotic resistance further makes it challenging for effective eradication. However, the mechanisms of immune evasion still remain obscure, and novel strategies should be developed to efficiently eliminate H. pylori infection in stomachs. Here we uncovered desirable anti-H. pylori effect of vitamin D3 both in vitro and in vivo, even against antibiotic-resistant strains. We showed that H. pylori can invade into the gastric epithelium where they became sequestered and survived in autophagosomes with impaired lysosomal acidification. Vitamin D3 treatment caused a restored lysosomal degradation function by activating the PDIA3 receptor, thereby promoting the nuclear translocation of PDIA3-STAT3 protein complex and the subsequent upregulation of MCOLN3 channels, resulting in an enhanced Ca release from lysosomes and normalized lysosomal acidification. The recovered lysosomal degradation function drives H. pylori to be eliminated through the autolysosomal pathway. These findings provide a novel pathogenic mechanism on how H. pylori can survive in the gastric epithelium, and a unique pathway for vitamin D3 to reactivate the autolysosomal degradation function, which is critical for the antibacterial action of vitamin D3 both in cells and in animals, and perhaps further in humans. Abbreviations: 1,25D3: 1α, 25-dihydroxyvitamin D3; ATG5: autophagy related 5; Baf A1: bafilomycin A; BECN1: beclin 1; CagA: cytotoxin-associated gene A; CFU: colony-forming unit; ChIP-PCR: chromatin immunoprecipitation-polymerase chain reaction; Con A: concanamycin A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTSD: cathepsin D; GPN: Gly-Phe-β-naphthylamide; H. pylori: Helicobacter pylori; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; MCOLN3: mucolipin 3; MCU: mitochondrial calcium uniporter; MOI: multiplicity of infection; NAGLU: N-acetyl-alpha-glucosaminidase; PDIA3: protein disulfide isomerase family A member 3; PMA: phorbol 12-myristate 13-acetate; PRKC: protein kinase C; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SS1: Sydney Strain 1; TRP: transient receptor potential; VacA: vacuolating cytotoxin; VD3: vitamin D3; VDR: vitamin D receptor.
Topics: Acetylglucosaminidase; Acid Phosphatase; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Calcium; Carrier Proteins; Cell Line; Cholecalciferol; Epithelial Cells; Helicobacter Infections; Helicobacter pylori; Humans; Lysosomes; Male; Mice, Inbred C57BL; Protein Disulfide-Isomerases; Receptors, Calcitriol; STAT3 Transcription Factor; Stomach; Transient Receptor Potential Channels; Cathelicidins
PubMed: 30612517
DOI: 10.1080/15548627.2018.1557835 -
Journal of the American Society of... May 2017Excessive fat intake contributes to the progression of metabolic diseases cellular injury and inflammation, a process termed lipotoxicity. Here, we investigated the...
Excessive fat intake contributes to the progression of metabolic diseases cellular injury and inflammation, a process termed lipotoxicity. Here, we investigated the role of lysosomal dysfunction and impaired autophagic flux in the pathogenesis of lipotoxicity in the kidney. In mice, a high-fat diet (HFD) resulted in an accumulation of phospholipids in enlarged lysosomes within kidney proximal tubular cells (PTCs). In isolated PTCs treated with palmitic acid, autophagic degradation activity progressively stagnated in association with impaired lysosomal acidification and excessive lipid accumulation. Pulse-chase experiments revealed that the accumulated lipids originated from cellular membranes. In mice with induced PTC-specific ablation of autophagy, PTCs of HFD-mice exhibited greater accumulation of ubiquitin-positive protein aggregates normally removed by autophagy than did PTCs of mice fed a normal diet. Furthermore, HFD-mice had no capacity to augment autophagic activity upon another pathologic stress. Autophagy ablation also exaggerated HFD-induced mitochondrial dysfunction and inflammasome activation. Moreover, renal ischemia-reperfusion induced greater injury in HFD-mice than in mice fed a normal diet, and ablation of autophagy further exacerbated this effect. Finally, we detected similarly enhanced phospholipid accumulation in enlarged lysosomes and impaired autophagic flux in the kidneys of obese patients compared with nonobese patients. These findings provide key insights regarding the pathophysiology of lipotoxicity in the kidney and clues to a novel treatment for obesity-related kidney diseases.
Topics: Animals; Autophagy; Diet, High-Fat; Kidney; Kidney Diseases; Lipid Metabolism; Lysosomes; Male; Mice; Palmitic Acid
PubMed: 27932476
DOI: 10.1681/ASN.2016070731 -
Molecules and Cells Aug 2023Lipofuscins are oxidized lipid and protein complexes that accumulate during cellular senescence and tissue aging, regarded as markers for cellular oxidative damage,...
Lipofuscins are oxidized lipid and protein complexes that accumulate during cellular senescence and tissue aging, regarded as markers for cellular oxidative damage, tissue aging, and certain aging-associated diseases. Therefore, understanding their cellular biological properties is crucial for effective treatment development. Through traditional microscopy, lipofuscins are readily observed as fluorescent granules thought to accumulate in lysosomes. However, lipofuscin granule formation and accumulation in senescent cells are poorly understood. Thus, this study examined lipofuscin accumulation in human fibroblasts exposed to various stressors. Our results substantiate that in glucose-starved or replicative senescence cells, where elevated oxidative stress levels activate autophagy, lipofuscins predominately appear as granules that co-localize with autolysosomes due to lysosomal acidity or impairment. Meanwhile, autophagosome formation is attenuated in cells experiencing oxidative stress induced by a doxorubicin pulse and chase, and lipofuscin fluorescence granules seldom manifest in the cytoplasm. As Torin-1 treatment activates autophagy, granular lipofuscins intensify and dominate, indicating that autophagy activation triggers their accumulation. Our results suggest that high oxidative stress activates autophagy but fails in lipofuscin removal, leaving an abundance of lipofuscin-filled impaired autolysosomes, referred to as residual bodies. Therefore, future endeavors in treating lipofuscin pathology-associated diseases and dysfunctions through autophagy activation demand meticulous consideration.
Topics: Humans; Lipofuscin; Aging; Cellular Senescence; Oxidative Stress; Lysosomes; Autophagy
PubMed: 37438887
DOI: 10.14348/molcells.2023.0019 -
Progress in Molecular and Subcellular... 2018In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating... (Review)
Review
In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca store, which can release Ca into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca stores play a role in shaping intracellular Ca signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed.
Topics: Animals; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Calcium; Endocytosis; Endoplasmic Reticulum; Endosomes; Humans; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Signal Transduction
PubMed: 30097775
DOI: 10.1007/978-3-319-96704-2_6 -
Cells Mar 2019The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to... (Review)
Review
The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.
Topics: Animals; Humans; Lysosomes; Mitophagy; Models, Biological; Phagosomes; Phosphorylation; rab GTP-Binding Proteins
PubMed: 30857122
DOI: 10.3390/cells8030224 -
Current Biology : CB Apr 2015
Topics: Humans; Lysosomes
PubMed: 25898096
DOI: 10.1016/j.cub.2015.02.027 -
Cellular Physiology and Biochemistry :... May 2021The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of... (Review)
Review
The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.
Topics: Animals; Cardiovascular Diseases; Endothelial Cells; Humans; Lysosomes; Myocytes, Smooth Muscle; Sphingolipids
PubMed: 34019755
DOI: 10.33594/000000373