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Autophagy Feb 2023In recent years, an increasing number of studies have started to investigate the roles of ions and ion channels in macroautophagy/autophagy. One finding is that calcium...
In recent years, an increasing number of studies have started to investigate the roles of ions and ion channels in macroautophagy/autophagy. One finding is that calcium regulates multiple stages of autophagy with lysosomal calcium release being important for autophagosome and lysosome fusion. MCOLN3/TRPML3, as a calcium-permeable channel that is located on both lysosomes and autophagosomes, has been suggested as an autophagy regulator and a candidate to provide the calcium for autophagic fusion, but how this channel is activated remains unclear. In a recent article, Kim et al. demonstrate that MCOLN3 is a PtdIns3P downstream effector, and the activation of its channel function is critical for autophagosome biogenesis.
Topics: Autophagosomes; Autophagy; Calcium; Calcium Channels; Lysosomes; Macroautophagy; Phosphatidylinositol Phosphates; Transient Receptor Potential Channels
PubMed: 36383451
DOI: 10.1080/15548627.2022.2148808 -
Autophagy May 2016The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles... (Review)
Review
The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.
Topics: Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Carrier Proteins; Humans; Phagosomes; Vacuoles
PubMed: 26986547
DOI: 10.1080/15548627.2016.1162364 -
Nature Communications Feb 2022Koolen-de Vries syndrome (KdVS) is a rare disorder caused by haploinsufficiency of KAT8 regulatory NSL complex subunit 1 (KANSL1), which is characterized by intellectual...
Koolen-de Vries syndrome (KdVS) is a rare disorder caused by haploinsufficiency of KAT8 regulatory NSL complex subunit 1 (KANSL1), which is characterized by intellectual disability, heart failure, hypotonia, and congenital malformations. To date, no effective treatment has been found for KdVS, largely due to its unknown pathogenesis. Using siRNA screening, we identified KANSL1 as an essential gene for autophagy. Mechanistic study shows that KANSL1 modulates autophagosome-lysosome fusion for cargo degradation via transcriptional regulation of autophagosomal gene, STX17. Kansl1 mice exhibit impairment in the autophagic clearance of damaged mitochondria and accumulation of reactive oxygen species, thereby resulting in defective neuronal and cardiac functions. Moreover, we discovered that the FDA-approved drug 13-cis retinoic acid can reverse these mitophagic defects and neurobehavioral abnormalities in Kansl1 mice by promoting autophagosome-lysosome fusion. Hence, these findings demonstrate a critical role for KANSL1 in autophagy and indicate a potentially viable therapeutic strategy for KdVS.
Topics: Abnormalities, Multiple; Animals; Autophagosomes; Cerebral Cortex; Chromosome Deletion; Chromosomes, Human, Pair 17; Disease Models, Animal; Female; Haploinsufficiency; HeLa Cells; Humans; Intellectual Disability; Isotretinoin; Lysosomes; Mice; Mice, Transgenic; Mitophagy; Neurons; Nuclear Proteins; Primary Cell Culture
PubMed: 35177641
DOI: 10.1038/s41467-022-28613-0 -
The Journal of Cell Biology Jun 2021Regulation of autophagy in neurons remains unclear. In this issue, Kulkarni et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202002084) show with elegant live...
Regulation of autophagy in neurons remains unclear. In this issue, Kulkarni et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202002084) show with elegant live imaging that in dendrites, but not in axons, autophagosome motility and function is regulated by synaptic activity.
Topics: Autophagosomes; Autophagy; Axons; Neurons; Synapses
PubMed: 33988696
DOI: 10.1083/jcb.202105008 -
The FEBS Journal May 2017Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the... (Review)
Review
Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence.
Topics: Adaptor Proteins, Vesicular Transport; Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Beclin-1; Class II Phosphatidylinositol 3-Kinases; Class III Phosphatidylinositol 3-Kinases; Endoplasmic Reticulum; Endosomes; Humans; Lysosomes; Membrane Microdomains; Models, Biological; Organelle Biogenesis; Phosphatidylinositol Phosphates; Second Messenger Systems
PubMed: 27973739
DOI: 10.1111/febs.13987 -
Autophagy Jun 2020Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed...
UNLABELLED
Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using C-labeled choline and C-magnetic resonance spectroscopy and western blotting, we show increased choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues.
ABBREVIATIONS
AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
Topics: Animals; Antineoplastic Agents; Autophagosomes; CHO Cells; Cell Line, Tumor; Choline; Choline-Phosphate Cytidylyltransferase; Cricetulus; Fibroblasts; Furans; Gene Knockout Techniques; Humans; Intracellular Membranes; Macroautophagy; Magnetic Resonance Spectroscopy; Mass Spectrometry; Metabolomics; Mice; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Phosphatidylcholines; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Pyrimidines; Vacuoles; bcl-2-Associated X Protein
PubMed: 31517566
DOI: 10.1080/15548627.2019.1659608 -
FEBS Letters Nov 2019Autophagy is widely considered as a housekeeping mechanism that enables cells to survive stress conditions and, in particular, nutrient deprivation. Autophagy begins... (Review)
Review
Autophagy is widely considered as a housekeeping mechanism that enables cells to survive stress conditions and, in particular, nutrient deprivation. Autophagy begins with the formation of the phagophore that expands and closes around cytosolic material and damaged organelles destined for degradation. The execution of this complex machinery is guaranteed by the coordinated action of more than 40 ATG (autophagy-related) proteins that control the entire process at different stages from the biogenesis of the autophagosome to cargo sequestration and fusion with lysosomes. Autophagosome biogenesis occurs at multiple intracellular sites, such as the endoplasmic reticulum (ER) and the plasma membrane. Soon after the formation of the phagophore, the nascent autophagosome progressively grows in size and ultimately closes by recruiting intracellular membranes. In this review, we focus on the contribution of three membrane sources - the ER, the ER-Golgi intermediate compartment, and the Golgi complex - to autophagosome biogenesis and expansion. We also highlight the interplay between the secretory pathway and autophagy in cells when nutrients are scarce.
Topics: Animals; Autophagosomes; Autophagy-Related Proteins; Endoplasmic Reticulum; Golgi Apparatus; Humans; Intracellular Membranes; Lysosomes
PubMed: 31603532
DOI: 10.1002/1873-3468.13637 -
The Journal of Biological Chemistry Nov 2023The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral...
The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.
Topics: Humans; Autophagy; DNA-Binding Proteins; HeLa Cells; Intercellular Signaling Peptides and Proteins; Lysosomes; Progranulins; Microtubule-Associated Proteins; Gene Expression Regulation; Extracellular Vesicles; Enzyme Inhibitors; Autophagosomes; Autophagy-Related Proteins
PubMed: 37739033
DOI: 10.1016/j.jbc.2023.105272 -
Autophagy Aug 20211-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and...
1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N635P: azido-STAR635P; NCy3: azido-cyanine 3; NpicCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.
Topics: Animals; Autophagosomes; Autophagy; Fibroblasts; Inflammasomes; Inflammation; Lysosomes; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Sphingolipids; Mice
PubMed: 32835606
DOI: 10.1080/15548627.2020.1804677 -
Journal of Molecular Biology May 2016Macroautophagy is an evolutionarily conserved dynamic pathway that functions primarily in a degradative manner. A basal level of macroautophagy occurs constitutively,... (Review)
Review
Macroautophagy is an evolutionarily conserved dynamic pathway that functions primarily in a degradative manner. A basal level of macroautophagy occurs constitutively, but this process can be further induced in response to various types of stress including starvation, hypoxia and hormonal stimuli. The general principle behind macroautophagy is that cytoplasmic contents can be sequestered within a transient double-membrane organelle, an autophagosome, which subsequently fuses with a lysosome or vacuole (in mammals, or yeast and plants, respectively), allowing for degradation of the cargo followed by recycling of the resulting macromolecules. Through this basic mechanism, macroautophagy has a critical role in cellular homeostasis; however, either insufficient or excessive macroautophagy can seriously compromise cell physiology, and thus, it needs to be properly regulated. In fact, a wide range of diseases are associated with dysregulation of macroautophagy. There has been substantial progress in understanding the regulation and molecular mechanisms of macroautophagy in different organisms; however, many questions concerning some of the most fundamental aspects of macroautophagy remain unresolved. In this review, we summarize current knowledge about macroautophagy mainly in yeast, including the mechanism of autophagosome biogenesis, the function of the core macroautophagic machinery, the regulation of macroautophagy and the process of cargo recognition in selective macroautophagy, with the goal of providing insights into some of the key unanswered questions in this field.
Topics: Autophagosomes; Autophagy; Organelle Biogenesis; Saccharomyces cerevisiae
PubMed: 26908221
DOI: 10.1016/j.jmb.2016.02.021