Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling

Significance The privileged interrelationship between mitochondria and the endoplasmic reticulum (ER) plays a key role in a variety of physiological functions, from lipid metabolism to Ca2+ signalling, and its modulation influences apoptotic susceptibility, mitophagy, and cellular bioenergetics. Among the several proteins known to influence ER–mitochondria interactions, mitofusin 2 (Mfn2) has been proposed to form a physical tether. In this study, we demonstrate that Mfn2 instead works as an ER–mitochondria tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles. Cells in which Mfn2 is ablated or reduced have an increased number of ER–mitochondria close contacts, potentiated Ca2+ transfer between the two organelles, and greater sensitivity to cell-death stimuli that implies mitochondria Ca2+ overload toxicity. The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER–mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca2+ transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca2+ overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER–mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER–mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.