Design of a bimolecular complementation assay to quantify mitochondrial fusion. (a) Schematic representation of the bimolecular complementation strategy used in the development of the mitochondrial cell free fusion assay. (b) Isolated mitochondria were incubated in the presence of 5 mg/mL placental cytosol and energy regeneration mixture for 30 min at either 37° in the absence (top panels) or presence (bottom panels) of apyrase. Confocal images of each reaction revealed the presence of respiring mitochondria labelled with MitofluorRed633 (middle panels). Fused mitochondria show complementation of Venus yellow fluorescent protein (YFP; right panels). Insets in the top panels show a 2.4× magnification of the area represented within the boxed region. Circled regions show mitochondria positive for Venus YFP and the arrow within the inset highlights a fused mitochondrion beside one that has not complemented the Venus fluorescence. Representative images are shown, scale bars, 10 μ. A Gaussian blur filter of 1 pixel was applied to these images. (c) Quantification of (b) taken from 200-350 mitochondria per condition. The data are the average of two independent experiments.
Schauss et al. BMC Biology 2010 8:100 doi:10.1186/1741-7007-8-100