Figure 7
Analyses of vMIA HHB and CBDII mutant clustering by MSIM and FLIM. HeLa cells were transiently transfected with vectors expressing (a) vMIA-EGFP, (b) vMIA-CBDII-EGFP or (c) vMIA-HHB-EGFP (green) as previously described36. The cells were co-transfected with only a mitochondrial marker (Mito-BFP, blue) (a,b), or with Mito-BFP and an ER marker (ER-RFP, red) (c). Cells were fixed and a single plane from an MSIM z-stack image showing distribution of vMIA-EGFP and mitochondrial and ER marker is shown. A zoom of regions marked by the white boxes in each image is shown below. (d) The percentages of mitochondria with clustered vMIA in HeLa cells expressing vMIA-EGFP (88.0 ± 3.7%, n = 5 cells) or vMIA-CBDII-EGFP (71.7 ± 4.8%, n = 6 cells). (d) The size of vMIA (mean 169.18, min: 110, median 167, max: 233, n = 50) and vMIA-CBDII (mean 164.55, min: 111, median 159, max: 230, n = 60) clusters were determined. p = 0.4024. The lines and red crosses indicate the medians and means, respectively. (f) The number of vMIA clusters in HeLa cells expressing vMIA-EGFP (mean 0.638, min: 0.194, median 0.562, max: 1.73, n = 50) or vMIA-CBDII-EGFP (mean = 0.563, min: 0.153, median 0.526, max: 1.312, n = 60) were determined (p = 0.3118). The line and red, cross mark indicate the median and mean, respectively. (g) vMIA-HHB and vMIA-CBDII are defective in dimerization as measured by FLIM. Shown are the fluorescence lifetime comparisons of vMIA-EGFP (τ = 3.10 ± 0.009 ns), vMIA-CBDII-EGFP (τ = 3.14 ± 0.007 ns) and vMIA-HHB-EGFP (τ = 3.2 ± 0.008 ns). *Represents p < 0.03, **represents p < 0.003, ****represents p = 0.0001.
