title: Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells.
abstract: Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol BS81TD, UK. t.macvicar@uni-koeln.de.
authors.forename: Thomas D B
authors.initials: TD
authors.lastname: MacVicar
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol BS81TD, UK. lilith.mannack@hotmail.co.uk.
authors.forename: Lilith V J C
authors.initials: LV
authors.lastname: Mannack
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol BS81TD, UK. rl14936@bristol.ac.uk.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Lees
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol BS81TD, UK. jon.lane@bristol.ac.uk.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Lane
mesh_terms: D001343:Autophagy; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D017480:Receptors, sigma / Q000037:antagonists & inhibitors* / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D000097605:Sigma-1 Receptor
chemical_list: D002118:Calcium; D053496:Inositol 1,4,5-Trisphosphate Receptors; D050918:Phosphatidylethanolamine N-Methyltransferase; D034741:RNA, Small Interfering; D017480:Receptors, sigma; D044767:Ubiquitin-Protein Ligases; D000097605:Sigma-1 Receptor; C494489:PEMT protein, human; C111567:parkin protein
keywords: ATG5; IP3Rs; LC3; Parkin; SIGMAR1; autophagy; calcium; mitophagy