Figure 1.
Visualization of ER-mitochondria tethering by electron microscopy.
Cells were fixed, embedded in Epon resin, sectioned and observed. (A) In WT MEFs expressing mitofusin-2, compartments continuous with ER cisternae were closely apposed to the mitochondrial outer membrane. Full arrowheads point to ribosomes attached to ER cisternae. Empty arrowheads designate the limits of the zone of intimate contact between ER and mitochondria. m: mitochondria. (B, C) Close ER-mitochondria apposition was also observed in mfn2 KO cells. In these cells, ER cisternae often appeared swollen (asterisks), but regions of tethering were still very thin.
Table 1.
ER-mitochondria contacts are increased in mfn2 KO cells.
Table 2.
No significant alteration in the overall surface of mitochondria in mfn2 KO cells.
Table 3.
ER cisternae are dilated in mfn2 KO cells.
Figure 2.
Complementation of mitofusin-2 KO cells.
Mfn2 KO cells were transfected with a plasmid driving the expression of Mitofusin-2 fused to GFP. Cells expressing moderate levels of fluorescence were sorted, re-grown, fixed and processed as described in the legend to Fig. 1. For each individual cell profile, the frequency of ER-mitochondria tethering (expressed as % of the total mitochondrial perimeter) was determined, as well as the average width of ER cisternae. Mock-transfected mfn2 KO cells (red circles) exhibited wider ER cisternae and more ER-mitochondria tethering than cells re-expressing Mitofusin-2 (green triangles). For each population of cells, the average and S.E.M. are indicated in black.
Table 4.
Expression of Mfn2-GFP complements the phenotype of mfn2 KO cells.
Figure 3.
Fluorescence analysis of ER and mitochondria.
WT (A) and mfn2 KO cells (B) were co-transfected with a GFP-coupled ER marker (green) and an RFP-coupled mitochondrial marker (red). After fixation, confocal images were acquired and used to quantify Manders' colocalization coefficients (Table 5). The arrowhead indicates a region of a mfn2 KO cell where the ER appears dilated. Bar: 10 µm.
Table 5.
Manders' colocalization coefficients in WT and mfn2 KO MEF cells.