Respiratory Chain Complexes in Dynamic Mitochondria Display a Patchy Distribution in Life Cells
Figure 3
Tagging of subunits of RC complexes does not interfere with mitochondrial localization, membrane-potential generation, respiration, or ultrastructure in stably transfected HeLa cells.
A–C. Expresssion of complex III subunit K fused to GFP (CIII-G) and labelling with the mitochondrial-specific dye MitoTracker Deep Red FM. C. The merged image shows clear co-localization indicating correct targeting of complex III subunit 10-GFP to mitochondria. D. The mitochondrial membrane potential in cells stable expressing the modified γ-subunit of complex V (CV-R-cells) is sustained. The merge (F) of the Complex V-γ-DsRed-signal (D) with the DASPMI-signal (E) shows the co-localization of the signal. G. Respiration of CV-R cells and cells stable expressing CIV-hAGT is not altered when endogenous substrate is oxidized, nor when substrates for the different RC complexes are added (complex I is supplied with NADH by the corresponding dehydrogenase feed with malate/glutamate, M/G; complex II is fueled with succinate, succ; and complex IV receives electrons from cytochrome c, which is reduced by TMPD in presence of ascorbate, A/T)[81]. Oxygen consumption rates derive from at n≥3 (CV-R: 4; HeLa: 3; CIV-hAGT: 5) independent cell preparations and measurements. H. Electron micrographs of mitochondria in HeLa cells. I. Electron micrographs of mitochondria in CV-R cells stably expressing the modified γ-subunit. Scale bars: 10 µm (A–C), 20 µm (D–F), 300 nm (H, I).