Maintenance of endoplasmic reticulum structural integrity is crucial for the execution of mitochondrial fission, outer membrane permeabilisation and apoptosis
Mateus Milani1, Gerald M. Cohen1,2 and Shankar Varadarajan1,2
Departments of 1Molecular and Clinical Cancer Medicine and 2Pharmacology, Institute of Translational Medicine, University of Liverpool, UK
Mitochondria are complex organelles responsible for diverse cellular mechanisms such as apoptosis, metabolism and bioenergetics. The mitochondrial fission machinery, comprising a dynamin-related GTPase, DRP-1, is crucial for the regulation of mitochondrial membrane dynamics and structural interconnectivity. Recent reports suggest that the tubular architecture of the endoplasmic reticulum (ER) could mark the constriction sites on the mitochondria to facilitate DRP-1-mediated mitochondrial fission. Since the ER exists as an elaborate network of tubes and sheets, we speculated whether modulating the expression levels of ER structuring proteins could alter mitochondrial architecture and function. To test this hypothesis, we genetically silenced the expression levels of key ER structuring proteins, namely Reticulon1 (RTN1), Reticulon4 (RTN4), Lunapark-1 (LNP1) and CLIMP-63 and assessed changes in mitochondrial structure and functions, following inducers of mitochondrial fission, such as A-1210477 (MCL-1 inhibitor) and CCCP (mitochondrial uncoupler). A genetic knockdown of DRP-1 prevented both A-1210477- and CCCP-mediated mitochondrial fission, and hence was used as a positive control. Downregulation of ER structuring proteins markedly decreased A-1210477-, but not CCCP-mediated mitochondrial fission. Moreover, knockdown of RTN1, RTN4 and CLIMP-63, but not LNP-1, prevented several hallmarks of apoptosis, ranging from mitochondrial outer membrane permeabilisation (characterised by the release of cytochrome c) to phosphatidylserine externalisation and caspase activation, following exposure to specific apoptotic stimuli. Interestingly, the role of ER shaping proteins in these pathways appeared to occur independently of OPA-1 proteolysis. Taken together, our results support the hypothesis that the integrity of mitochondrial fission machinery and ER membranes are required for the onset of mitochondrial fission, outer membrane permeabilisation and apoptosis, thus highlighting the importance of ER structural proteins and ER structural integrity in the efficient execution of apoptosis.