Life cell quantification of mitochondrial membrane potential at the single organelle level

Abstract

Mitochondrial membrane potential (Dw) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Dw in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is sequestered by mitochondria according to their Dw. Cells were visualized by videomicroscopy and the acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence. Following validation, our approach revealed that mitochondria with different Dw coexisted within the same cell. Furthermore, our method allowed reproducible detection of small (\10%) reductions in TMRM intensity induced by the complex III inhibitor antimycin A. Mitochondrial uncoupling by p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) greatly reduced mitochondrial TMRM fluorescence. Under these conditions faithful mask calculation and TMRM intensity analysis were still possible using a mitochondria-targeted green fluorescence protein (mitoAcGFP1), expressed in the cells using baculoviral transfection. ' International Society for Analytical CytologyKey terms fibroblasts; TMRM; AcGFP1; image processing and analysis MITOCHONDRIA actively maintain a highly negative potential across their inner membrane (Dw), which is essential for mitochondrial function and cell viability. This potential is maintained by four protein complexes (CI-CIV) of the mitochondrial electron transport chain (ETC) that, together with the F 0 /F 1 -ATP-synthase (CV), constitute the oxidative phosphorylation (OXPHOS) system (1). Mitochondria are fuelled by pyruvate and fatty acids, which are used as carbon sources for the tricarboxylic acid cycle in the mitochondrial matrix. The products of this cycle, NADH and FADH 2 , subsequently feed electrons into the ETC at CI and CII, respectively. At three locations within the chain, CI, CIII, and CIV, the electron transport is used to expel protons from the matrix space to the intermembrane space. The H 1 ejection results in the establishment of an electrochemical H 1 gradient, which consists of a chemical (DpH) and electrical (Dw) component. At CV, protons are allowed to flow back into the mitochondrial matrix to drive the synthesis of ATP from ADP and inorganic phosphate (P i ). In intact respiring mitochondria, the ETC and ATP synthesis are efficiently coupled. Importantly, a large number of diseases have been linked to mitochondrial dysfunction, including age-related neurological diseases, cardiovascular disease, cancer and diabetes (reviewed in Refs. 2, 3).Since Dw is regarded as a key indicator of mitochondrial health and metabolic activity, the accurate determination of this parameter in intact cells is important to address many research questions. Most assays for monitoring Dw in living cells

Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient

“…Previous work indicated some heterogeneity in the respiratory activities of the mitochondria of an individual cell (21)(22)(23)(24). Indeed, the DiOC 6 staining of living cells, as shown in Figs.…”

Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution

“…Following this, the described heterogeneous spatiotemporal organization of OXPHOS complexes -trapped in the cristae microcompartment -provides an explanation for mitochondrial heterogeneity. Therefore, zones of different membrane potential within single mitochondria (Bereiter-Hahn and Voeth, 1998;Collins et al, 2002;Distelmaier et al, 2008;Wikstrom et al, 2009) could be assigned to local accumulation of functional compromised OXPHOS complexes that are not or only slowly exchanged by remixing. For the future, it would be exciting to identify and characterize these zones more precisely and correlate local membrane potential with the actual distribution and functionality of the present OXPHOS complexes.…”

Trolox-Sensitive Reactive Oxygen Species Regulate Mitochondrial Morphology, Oxidative Phosphorylation and Cytosolic Calcium Handling in Healthy Cells

“…Biopsies were performed following informed consent and according to the relevant Institutional Review Boards. Fibroblasts were cultured as previously described (6). CHO cells, HeLa cells, and immortalized MEFs were cultured as described in the Supplementary Materials and Methods.…”

“…Mitochondrial tetramethyl rhodamine methyl ester (TMRM; Invitrogen) staining was used to estimate Dw; staining with TMRM or rhodamine 123 (Invitrogen) was used to quantify mitochondrial morphology. The TMRM and R123 approaches were previously described in detail (6,24,26). Measurements of mitochondrial NAD(P)H autofluorescence were carried out as described earlier (43).…”