title: Modulation of intracellular calcium homeostasis blocks autophagosome formation.
abstract: Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.
authors: Centre for Molecular Medicine Norway (NCMM); Nordic EMBL Partnership; University of Oslo, Norway.
authors: Nikolai
authors: N
authors: Engedal
authors: Department of Biochemistry; Institute of Basic Medical Sciences; Faculty of Medicine; University of Oslo, Norway.
authors: Maria L
authors: ML
authors: Torgersen
authors: Centre for Molecular Medicine Norway (NCMM); Nordic EMBL Partnership; University of Oslo, Norway.
authors: Ingrid J
authors: IJ
authors: Guldvik
authors: Centre for Molecular Medicine Norway (NCMM); Nordic EMBL Partnership; University of Oslo, Norway.
authors: Stefan J
authors: SJ
authors: Barfeld
authors: Autophagy Laboratory; Department of Molecular Biology; Interfaculty Institute for Cell Biology; Faculty of Science; Eberhard Karls University; Tuebingen, Germany.
authors: Daniela
authors: D
authors: Bakula
authors: Department of Cell Biology; Institute for Cancer Research; Norwegian Radium Hospital; Oslo University Hospital; Nydalen, Oslo Norway.
authors: Frank
authors: F
authors: Sætre
authors: Department of Cell Biology; Institute for Cancer Research; Norwegian Radium Hospital; Oslo University Hospital; Nydalen, Oslo Norway.
authors: Linda K
authors: LK
authors: Hagen
authors: MannKind Corporation; Valencia, CA USA.
authors: John B
authors: JB
authors: Patterson
authors: Autophagy Laboratory; Department of Molecular Biology; Interfaculty Institute for Cell Biology; Faculty of Science; Eberhard Karls University; Tuebingen, Germany.
authors: Tassula
authors: T
authors: Proikas-Cezanne
authors: Department of Cell Biology; Institute for Cancer Research; Norwegian Radium Hospital; Oslo University Hospital; Nydalen, Oslo Norway.
authors: Per O
authors: PO
authors: Seglen
authors: Department of Biochemistry; Institute of Basic Medical Sciences; Faculty of Medicine; University of Oslo, Norway.
authors: Anne
authors: A
authors: Simonsen
authors: Centre for Molecular Medicine Norway (NCMM); Nordic EMBL Partnership; University of Oslo, Norway; Department of Cancer Prevention and Department of Urology; Oslo University Hospitals; Oslo, Norway.
authors: Ian G
authors: IG
authors: Mills
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D019284:Thapsigargin; D002118:Calcium
keywords: A23187; ER stress; autophagosome formation; autophagy; calcium; long-lived protein degradation; sequestration; thapsigargin