Riccardo Filadi, Elisa Greotti, Gabriele Turacchio,
Alberto Luini, Tullio Pozzan and
Interactions between mitochondria and the endoplasmic reticulum (ER) have been studied a lot and have important physiological functions (e.g. lipid metabolism and Ca²⁺ signalling). Several proteins are involved in regulating the interactions between the ER and mitochondria. The mitochondrial fusion protein Mfn2 has been proposed to be involved in the physical tethering. Quantitative EM analysis, however, challenges this view. In this paper they find that Mfn2 actually prevents an excessive proximity between mitochondria and ER and that cells with reduced Mfn2 show more ER-mitochondria contact points which causes a greater sensitivity to death stimuli.
EM analysis was done in mouse embryonic fibroblasts with knocked down (KD) or knocked out (KO) Mfn2. An increase in the number of close (<15 nm) mitochondria-ER contacts in Mfn2 KO cells and Mfn2 KD cells. When using confocal miscroscopy, however, a decrease in the overlapping area between ER and mitochondria was seen in mutant cells compared to wild-type cells.
The authors explain this discrepancy by arguing that Mfn2 ablation causes mitochondria to swell. EM measures the number and size of contacts between perimeters of the organelles, whereas fluorescence microscopy usually uses luminal fluorescent proteins and measures overlapping areas/volumes. Mitochondrial swelling makes its area increase more than the perimeter which could results in an apparent decrease in ER-mitochondria tethering when using fluorescent microscopy.
More ER-mitochondria contact points should lead to more calcium uptake by mitochondria. However, mitochondria took up less calcium in Mfn2 KO cells compared to wildtype cells. The authors explain this by arguing that expression levels of the mitochondrial calcium uniporter (MCU) were reduced by about 50% in Mfn2 KO cells. The reduced calcium uptake by mitochondria is thus suggested to be a cause of a reduction in MCU expression level, rather than a change in the distance between ER and mitochondria.
A comparison between wildtype and Mfn2 KD cells showed that Mfn2 KD cells show similar ATP levels and respiration levels but lowered membrane potential compared to wildtype. The increase in contact points in Mfn2 KD cells led to a greater sensitivity to apoptotic stimuli.