Figure 8. Cytosolic mitofusin blocks mitochondrial tethering by mitofusin proline variants.

(A) Schematic of mitochondria tethered by mitofusin trans complexes (left) and mitochondria with mitofusin trans complexes blocked by interaction with the cytosolic variant of mitofusin (F646D) (right). (B) Representative images of Flp-In TREx HEK293 transfected with eGFP, eGFP-Mfn1^F646D, or eGFP-Mfn1^F646D-S329P and expressing either Mfn1^WT-FLAG or Mfn1^S329P-FLAG after incubation with 0.2 μg/ml TET for 4 h. Mitochondria were labeled with MitoTracker Red CMXRos, and nuclei were labeled with NucBlue and visualized by live-cell fluorescence microscopy. Images represent maximum intensity projections. Scale bar = 5 μm. (B, C) Quantification of the mitochondrial morphology in the cell lines described in (B). Error bars represent mean ± SEM from n = 3 separate blinded experiments (≥100 cells per experiment).