Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC50 values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

[1]  B. Ames,et al.  Mitochondrial Decay in Aging: Reversal through Supplementation of Acetyl‐l‐Carnitine and N‐tert‐Butyl‐α‐phenyl‐nitrone a , 1998, Annals of the New York Academy of Sciences.

[2]  L. Marshall,et al.  A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells. , 2001, Analytical biochemistry.

[3]  J. Houštěk,et al.  Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin , 1999, Bioscience reports.

[4]  L. Loew,et al.  Physiological cytosolic Ca2+ transients evoke concurrent mitochondrial depolarizations. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  N. Howell.  Human mitochondrial diseases: answering questions and questioning answers. , 1999, International review of cytology.

[6]  R C Scaduto,et al.  Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. , 1999, Biophysical journal.

[7]  M. Klingenberg,et al.  Structure and function of the uncoupling protein from brown adipose tissue. , 1999, Biochimica et biophysica acta.

[8]  R. Weindruch,et al.  Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes. , 1998, American journal of physiology. Endocrinology and metabolism.

[9]  A. Schapira,et al.  Mitochondrial myopathies and encephalomyopathies , 1999, European journal of clinical investigation.

[10]  O. Larsson,et al.  Glucose-induced Oscillations in Cytoplasmic Free Ca2+Concentration Precede Oscillations in Mitochondrial Membrane Potential in the Pancreatic β-Cell* , 2001, The Journal of Biological Chemistry.

[11]  M. Klingenberg.  Mechanism and evolution of the uncoupling protein of brown adipose tissue. , 1990, Trends in biochemical sciences.

[12]  W. Tatton,et al.  Mitochondrial Membrane Potential in Aging Cells , 2001, Neurosignals.

[13]  T. Lampidis,et al.  Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells. , 1985, The Journal of biological chemistry.

[14]  M. Ward,et al.  Mitochondrial membrane potential and neuronal glutamate excitotoxicity: mortality and millivolts , 2000, Trends in Neurosciences.

[15]  P. Mitchell.  Vectorial chemiosmotic processes. , 1977, Annual review of biochemistry.

[16]  D. Nicholls,et al.  Thermogenic mechanisms in brown fat. , 1984, Physiological reviews.

[17]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[18]  C. Gabriel,et al.  Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry. , 1997, Cytometry.

[19]  P. O'Brien,et al.  Cancer Chemoprevention and Apoptosis Mechanisms Induced by Dietary Polyphenolics , 2000, Drug metabolism and drug interactions.

[20]  B. Frenguelli,et al.  Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells , 2000, The Journal of Neuroscience.

[21]  G. Kroemer,et al.  Quantitation of mitochondrial transmembrane potential in cells and in isolated mitochondria. , 2000, Methods in enzymology.

[22]  J. Prehn,et al.  Mitochondrial Depolarization Is Not Required for Neuronal Apoptosis , 1999, The Journal of Neuroscience.

[23]  G. Kroemer,et al.  Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins , 1998, Oncogene.

[24]  Y. Tsujimoto,et al.  Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Wallace.  Mitochondrial diseases in man and mouse. , 1999, Science.

[26]  D L Farkas,et al.  Simultaneous imaging of cell and mitochondrial membrane potentials. , 1989, Biophysical journal.

[27]  L. Leybaert,et al.  Mitochondrial Transmembrane Potential Changes Support the Concept of Mitochondrial Heterogeneity During Apoptosis , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[28]  M. Duchen.  Mitochondria and calcium: from cell signalling to cell death , 2000, The Journal of physiology.

[29]  J. Lemasters,et al.  Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader. , 2001, Analytical biochemistry.

[30]  D. Choi,et al.  Mediation of neuronal apoptosis by enhancement of outward potassium current. , 1997, Science.

[31]  G. Kroemer,et al.  Mitochondria as regulators of apoptosis: doubt no more. , 1998, Biochimica et biophysica acta.

[32]  L. B. Chen,et al.  Mitochondrial membrane potential in living cells. , 1988, Annual review of cell biology.

[33]  I. Nelson,et al.  Genetics and molecular pathogenesis of mitochondrial respiratory chain diseases , 1999, Cellular and Molecular Life Sciences CMLS.

[34]  W. Tatton,et al.  Reduced mitochondrial membrane potential and altered responsiveness of a mitochondrial membrane megachannel in p53-induced senescence. , 1999, Biochemical and biophysical research communications.

[35]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .