Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F0 during ATP synthesis

It has been suggested that ischemic renal proximal tubular cell injury is mediated by an increase in cytosolic calcium concentrations ((Ca2+)i). However, measurements of (Ca2+)i in rat or rabbit proximal tubules exposed to hypoxia or anoxia have yielded ambiguous results. This study explored the possibility that the severity of oxygen deprivation and the energy state of the mitochondria are important determinants of (Ca2+)i. To this end, (Ca2+)i (measured with fura-2) and the mitochondrial membrane potential (measured with rhodamine 123) were studied simultaneously in individual rat proximal tubules in hypoxic and anoxic conditions. (Ca2+)i did not change during hypoxia, but increased rapidly during anoxia. Increases in (Ca2+)i were only observed in parallel with a decrease of rhodamine 123 fluorescence, which indicates a collapse of the mitochondrial membrane potential. The increase in (Ca2+)i during anoxia was prevented by incubating the tubules in a low Ca2+ medium, which did not interfere with the collapse of the mitochondrial membrane potential. Both hypoxic and anoxic incubation led to cell death, as assessed by the fluorescent dye propidium iodide. These results clearly demonstrate that the level of oxygen deprivation is critical in determining changes in (Ca2+)i. Because cell damage occurred in both hypoxic and anoxic conditions. It was concluded that an increase in (Ca2+)i is not a necessary prerequisite for the development of ischemic cell injury.

Download Full-text

Time-dynamics of mitochondrial membrane potential reveal an inhibition of ATP synthesis in mitosis

10.1101/772244 ◽

2019 ◽

Cited By ~ 2

Author(s):

Joon Ho Kang ◽

Georgios Katsikis ◽

Max A. Stockslager ◽

Daniel Lim ◽

Michael B. Yaffe ◽

...

Keyword(s):

Cell Division ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Atp Synthesis ◽

Electrical Circuit ◽

Time Dynamics ◽

Delay Cell ◽

Transport Chain ◽

A Cell

AbstractThe energetic demands of a cell are believed to increase during mitosis 1–7. As cells transit from G2 into mitosis, mitochondrial electron transport chain (ETC) activity increases 4,8,9, and cellular ATP levels progressively decrease until the metaphase-anaphase transition 3,7,10, consistent with elevated consumption. The rates of ATP synthesis during mitosis, however, have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytes and demonstrate that cyclin-dependent kinase 1 (CDK1) activity causes mitochondrial hyperpolarization from G2/M until the metaphase-anaphase transition. By using an electrical circuit model of mitochondria, we quantify the time-dynamics of mitochondrial membrane potential under normal and perturbed conditions to extract mitochondrial ATP synthesis rates in mitosis. We found that mitochondrial ATP synthesis decreases by approximately 50 % during early mitosis, when CDK1 is active, and increases back to G2 levels during cytokinesis. Consistently, acute inhibition of mitochondrial ATP synthesis failed to delay cell division. Our results provide a quantitative understanding of mitochondrial bioenergetics in mitosis and challenge the traditional dogma that cell division is a highly energy demanding process.

Download Full-text

Mitochondrial membrane potential regulation is independent of c-fos expression

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y99-007 ◽

1999 ◽

Vol 77 (3) ◽

pp. 195-203

Author(s):

Roger A Moorehead ◽

Gurmit Singh

Keyword(s):

Membrane Potential ◽

Ovarian Carcinoma ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Membrane Potentials ◽

Rhodamine 123 ◽

Parental Line ◽

Rat Fibroblasts ◽

Human Ovarian Carcinoma ◽

Fos Expression

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.Key words: c-fos, mitochondria, membrane potential, rhodamine 123 (Rh123), lipophilic cations.

Download Full-text

Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division

Nature Communications ◽

10.1038/s41467-020-18769-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Joon Ho Kang ◽

Georgios Katsikis ◽

Zhaoqi Li ◽

Kiera M. Sapp ◽

Max A. Stockslager ◽

...

Keyword(s):

Cell Division ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Leukemia Cell ◽

Atp Synthesis ◽

Electrical Circuit ◽

Lymphocytic Leukemia ◽

Time Dynamics ◽

Lymphocytic Leukemia Cell

Abstract The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

Download Full-text

Mitochondrial Regulation of Calcium in the Avian Cochlear Nucleus

Journal of Neurophysiology ◽

10.1152/jn.1997.78.4.1928 ◽

1997 ◽

Vol 78 (4) ◽

pp. 1928-1934 ◽

Cited By ~ 10

Author(s):

Sam P. Mostafapour ◽

Edward A. Lachica ◽

Edwin W Rubel

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Cochlear Nucleus ◽

Mitochondrial Membrane ◽

Atp Synthesis ◽

Cytosolic Calcium ◽

Mitochondrial Regulation ◽

Mitochondrial Number ◽

And Function

Mostafapour, Sam P., Edward A. Lachica, and Edwin W Rubel. Mitochondrial regulation of calcium in the avian choclear nucleus. J. Neurophysiol. 78: 1928–1934, 1997. The role of mitochondria and the endoplasmic reticulum in buffering [Ca2+]i in response to imposed calcium loads in neurons of the chick cochlear nucleus, nucleus magnocellularis (NM), was examined. Intracellular calcium concentrations were measured using fluorometric videomicroscopy. After depolarization with 125 mM KCl, NM neurons demonstrate an increase in [Ca2+]i that returns to near-basal levels within 6 min. Addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) dissipated the mitochondrial membrane potential, as evidenced by increased fluorescence when cells were loaded with rhodamine-123. Two micromolar CCCP had minimal effect on baseline [Ca2+]i. However, 2 or 10 μM CCCP interfered with the ability of NM cells to buffer [Ca2+]i in response to KCl depolarization without significantly affecting peak [Ca2+]i. Oligomycin also interfered with postdepolarization regulation of [Ca2+]i, but blocked late (7–8 min postdepolarization) increases in [Ca2+]i caused by CCCP. Thapsigargin had no effect on baseline, peak, or postdepolarization [Ca2+]i in NM cells. These results suggest that normal mitochondrial membrane potential and ATP synthesis play an important role in buffering [Ca2+]i in response to imposed calcium loads in NM neurons. Furthermore, the endoplasmic reticulum does not appear to play a significant role in either of these processes. Thus increases in mitochondrial number and function noted in NM cells after deafferentation may represent an adaptive response to an increased cytosolic calcium load.

Download Full-text

Cadmium-Induced Mitochondrial Membrane-Potential Dissipation Does Not Necessarily Require Cytosolic Oxidative Stress: Studies Using Rhodamine-123 Fluorescence Unquenching

Toxicological Sciences ◽

10.1093/toxsci/kfh015 ◽

2004 ◽

Vol 77 (2) ◽

pp. 299-306 ◽

Cited By ~ 38

Author(s):

J.-S. Bolduc

Keyword(s):

Oxidative Stress ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Rhodamine 123 ◽

Stress Studies

Download Full-text

SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential

10.1101/2021.01.03.425119 ◽

2021 ◽

Author(s):

Nadezda A. Brazhe ◽

Evelina I. Nikelshparg ◽

Adil A. Baizhumanov ◽

Vera G. Grivennikova ◽

Anna A. Semenova ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Conformational Changes ◽

Mitochondrial Membrane ◽

Atp Synthesis ◽

Surface Enhanced Raman Spectroscopy ◽

Complex Iii ◽

Label Free ◽

Inner Mitochondrial Membrane ◽

The Impact

AbstractCytochrome c is an essential component of the electron transport chain (ETC), which regulates respiratory chain activity, oxygen consumption, and ATP synthesis. But the impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. Here we describe a label-free tool that identifies conformational changes in cytochrome c heme and elucidates their function. We verify that molecule bond vibrations assessed by surface-enhanced Raman spectroscopy (SERS) is a reliable indicator of the planar heme configuration during activation of ETC and decrease in inner mitochondrial membrane potential. The planar conformation of cytochrome c heme enables its optimal orientation towards the heme of cytochrome c1 in complex III. This ensures a faster electron transfer, which is important during ETC speed-ups and acceleration of ATP synthesis. The ability of our tool to track mitochondrial function opens wide perspectives on cell bioenergetics.

Download Full-text