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dc.contributor.authorAndrejeva, G
dc.contributor.authorGowan, S
dc.contributor.authorLin, G
dc.contributor.authorWong Te Fong, A-CL
dc.contributor.authorShamsaei, E
dc.contributor.authorParkes, HG
dc.contributor.authorMui, J
dc.contributor.authorRaynaud, FI
dc.contributor.authorAsad, Y
dc.contributor.authorVizcay-Barrena, G
dc.contributor.authorNikitorowicz-Buniak, J
dc.contributor.authorValenti, M
dc.contributor.authorHowell, L
dc.contributor.authorFleck, RA
dc.contributor.authorMartin, L-A
dc.contributor.authorKirkin, V
dc.contributor.authorLeach, MO
dc.contributor.authorChung, Y-L
dc.date.accessioned2019-09-17T10:36:39Z
dc.date.issued2020-06
dc.identifier.citationAutophagy, 2020, 16 (6), pp. 1044 - 1060
dc.identifier.issn1554-8627
dc.identifier.urihttps://repository.icr.ac.uk/handle/internal/3345
dc.identifier.eissn1554-8635en_US
dc.identifier.doi10.1080/15548627.2019.1659608en_US
dc.description.abstractMacroautophagy/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 <sup>13</sup>C-labeled choline and <sup>13</sup>C-magnetic resonance spectroscopy and western blotting, we show increased <i>de novo</i> 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 <i>de novo</i> 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 <i>de novo</i> 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.
dc.formatPrint-Electronic
dc.format.extent1044 - 1060
dc.languageeng
dc.language.isoeng
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.title<i>De novo</i> phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.
dc.typeJournal Article
dcterms.dateAccepted2019-08-12
rioxxterms.versionofrecord10.1080/15548627.2019.1659608
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0
rioxxterms.licenseref.startdate2020-06en_US
rioxxterms.typeJournal Article/Review
dc.relation.isPartOfAutophagy
pubs.issue6
pubs.notesNot known
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Breast Cancer Research
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Breast Cancer Research/Endocrinology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Cancer Pharmacology & Stress Response (CPSR)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Cancer Pharmacology & Stress Response (CPSR)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Clinical Pharmacology & Trials (including Drug Metabolism & Pharmacokinetics Group)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Molecular Pathology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Molecular Pathology/Endocrinology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Radiotherapy and Imaging
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Radiotherapy and Imaging/Magnetic Resonance
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Breast Cancer Research
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Breast Cancer Research/Endocrinology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Cancer Pharmacology & Stress Response (CPSR)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Cancer Pharmacology & Stress Response (CPSR)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Clinical Pharmacology & Trials (including Drug Metabolism & Pharmacokinetics Group)
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Molecular Pathology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Molecular Pathology/Endocrinology
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Radiotherapy and Imaging
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Radiotherapy and Imaging/Magnetic Resonance
pubs.publication-statusPublished
pubs.volume16en_US
pubs.embargo.termsNot known
icr.researchteamCancer Pharmacology & Stress Response (CPSR)en_US
icr.researchteamClinical Pharmacology & Trials (including Drug Metabolism & Pharmacokinetics Group)en_US
icr.researchteamEndocrinologyen_US
icr.researchteamMagnetic Resonanceen_US
dc.contributor.icrauthorRaynaud, Florenceen
dc.contributor.icrauthorChung, Yuen-Lien
dc.contributor.icrauthorMartin, Lesley-Annen
dc.contributor.icrauthorParkes, Harolden
dc.contributor.icrauthorKirkin, Vladimiren


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