Figure 4: Gβ2 requires Mfn1 for its effect on mitochondrial dynamics.

(a) Representative images of wild type or Mfn1^−/− and/or Mfn2^−/− MEF cells transfected with Flag-tagged Gβ2. Mitochondria in cells were stained with MitoTracker Red and Flag- Gβ2 were immunostained with a Flag-specific antibody. Scale bar, 10 μm. (b) Mitochondria in Mfn2^−/− cells, and Mfn1^−/− cells are shown in the representative images of Mfn1^−/− or Mfn2^−/− MEFs transfected with Gβ2 shRNA (red). The box area A (tranfected cell) and B (untransfectd cell) in the left panel show the different mitochondrial size at higher magnifications. Scale bar, 10 μm. (c) The quantification of mitochondrial size scored by Image J (Wayne Rasband, NIH). Filled box represents mitochondrial size in Mfn2^−/− MEFs and open box represents in Mfn2^−/− MEFs transfected with Gβ2 shRNA. The data represent the mean s.d., n=10–15. Differences in the mean mitochondrial average area between mfn2^−/− MEFs and mfn2^−/− MEFs with Gβ2 shRNA were statistically significant at *P<0.05. (d) Mfn1-YFP was co-transfected into HeLa cells with either Flag- Gβ2 ( , black) or Gβ2 shRNA (, red) and the mitochondrial Mfn1 mobility was measured by FRAP. Fis1 (, green) was used as a control of fast membrane mobility as previously reported²⁸ and Mfn1 alone ( , blue) was used as baseline control. Each line represents the mean of ≥20 measurements. (e) The fluorescence recovery of Mfn1-YFP in HeLa cells and the cells co-transfected with either Flag-Gβ2 or Gβ2 shRNA was measured by FRAP. Differences in the mean fluorescence recovery between Mfn1-YFP in HeLa cells and Mfn1-YFP in HeLa cells co-transfected with Flag-Gβ2, or with Gβ2 shRNA were statistically significant *P<0.05 and **P<0.005; Error bars represent the s.e.m., n=21. (f) Mobility of Mfn1-GFP, or Mfn1-GFP (Δ241–350), Mfn1-GFP (Δ1–387) in HeLa cells and the cells co-transfected with DsRed-Gβ2 shRNA was measured by FRAP. The fluorescence recovery of Mfn1-GFP, Mfn1-GFP (Δ241–350) and Mfn1-GFP (Δ1–387) in HeLa cells and the cells co-transfected with Gβ2 shRNA was measured by FRAP. *P<0.05 and NS means nonsignificant. Data represent the mean s.e.m., n=20.