Hybrid mitochondria following mitochondrial fusion display a patterned distribution of RC complexes.

Ongoing fusion and fission of mitochondria subsequent to cell fusion caused the generation of hybrid mitochondria with a mixed population of RC complexes. A. Appearance of mitochondria with CIII-R and CII-g 5d after coexpression. Lower part: Line plot of CIII-R and CII-G fluorescence intensities along the longitudinal axis of a single mitochondrion, indicated with a white arrow head in A. The corresponding Pearson's cross correlation coefficient is 0.89 calculated from the fluorescence distribution in a single mitochondrion as depicted in the lower part. B. CV-R and CV-G distribution in hybrid mitochondria 5 h after cell fusion. The corresponding Pearson coefficient is 0.49 calculated from the line plot in the lower part. C. CV-R and CIII-G fusion and analysis 4 h later. Lower part: Line plot of CIII-G and CV-R fluorescence intensities along the longitudinal axis of a single mitochondrion, indicated with a white arrow head in C. The corresponding Pearson's cross correlation coefficient is 0.38. D. CI-G and CV-R in mitochondria 4.5 h after cell fusion. Lower part: Line plot of CI-G and CV-R fluorescence intensities along the longitudinal axis of a single mitochondrion, indicated with a white arrow. The corresponding Pearson's cross correlation coefficient is 0.49. Some fragmented mitochondria display only CV-R fluorescence, because they did not fuse with CI-G-mitochondria (arrow). E. CI-G and CIII-R fluorescence distribution in mitochondria 5 h after fusion. The calculated Pearson for the correlation of the fluorescence distribution is 0.62. F. CI-G and mtDsRed fluorescence distribution in mitochondria in cells co-expressing both plasmids. The corresponding Pearson's cross correlation coefficient is 0.66. Scales bars 1 µm  = 30−40 pixel (A, C, D E), 1 µm  = 108 pixel (B), 1 µm  = 134 pixel (F). After recording z-stacks, the images were deconvolved by ®Autoquant software before quantitative analysis. G. Comparison of fluorescence distribution in CI-G and CV-R fused and co-expressing cells under different conditions reveal an increase in homogeneity by time due to ongoing fusion dynamics. 24 h after cell fusion the mixing of RC is complete as proved by the comparison with co-expressing cells (n≥3 independent cell assays for 4 h, 24 h CHX and coexpression; n = 2 cell assay for 23 h no CHX). H. Complex II mixes better with other complexes in co-expressing cells than complex I and V, resp. (students t-test, significance level p = 0.05) I.J. Cells with red and green fluorescent RC complexes were fused and fluorescence distribution of RC complexes in hybrid mitochondria 4–5 h after fusion as well as in mitochondria from co-expressing cells was determined. The cross-correlation of the two fluorescence channels, calculated according to Pearson, was taken as a measure for the homogeneity of RC complex distribution. For each combination at least 15 heterokaryons from 3 different independent cell fusion/co-expression assays were investigated. The data are shown as mean ± s.e.m (n≥3). Significance of differences from the relevant controls was calculated by Students' t test.