Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells.

Thomas Macvicar,Jon Lane,Robert Lees,Lilith Mannack

doi:10.3390/ijms160613356

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.

Highlights

Autophagy is a highly conserved catabolic process encompassing distinct pathways for the delivery of cytoplasmic material to the lysosome for degradation and recycling
The above studies suggested that Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria might be a key step in the acute mitophagy response, so in order to test whether uptake of Ca2+ into mitochondria influences mitophagy we focused on the mitochondrial Ca2+ uniporter (MCU [45])
The finding that the early autophagosome assembly machinery is focused at ER-mitochondrial contact sites, and that stress-induced autophagosomes originate at or close to these sites [8], supports a persuasive argument that ER and mitochondrial homeostasis combine to coordinate both stress-induced autophagy and mitophagy responses to drive the selective engulfment of mitochondria that are in physical contact with the ER

Summary

Introduction

Autophagy is a highly conserved catabolic process encompassing distinct pathways for the delivery of cytoplasmic material to the lysosome for degradation and recycling. Important studies using chicken DT40 cells lacking all 3 IP3R isoforms which can be rescued using channel mutant IP3Rs, suggested that altered baseline autophagy in the absence of IP3Rs is more likely linked to changes in Ca2+ regulated mitochondrial bioenergetics [28,29], rather than being due to the physical association between IP3Rs and Beclin-1 that normally occurs at contact sites between mitochondria and the ER [30]. We have used siRNA to screen for involvement of regulators of ER and mitochondrial structural dynamics and ER-mitochondrial contact to better understand the relationships between ER and mitochondria during starvation mediated autophagy and Parkin-dependent mitophagy. Using hTERT-immortalised human retinal pigment epithelial cells (RPE1) stably expressing YFP-Parkin [2], or either mCherry-GFP-LC3B or GFP-ATG5, we have identified several potentially important players in ER-mitochondrial lipid exchange, Ca2+ transfer and ER homeostasis in both autophagy and mitophagy

Results

Discussion

Chemicals and Antibodies

Cell Culture and Cell-Lines

Transfection and Viral Transduction

Microscopy