A High-Throughput Assay for Mitochondrial Membrane Potential in Permeabilized Yeast Cells

2001 ◽  
Vol 293 (2) ◽  
pp. 269-276 ◽  
Author(s):  
Ellyn Farrelly ◽  
M.Catherine Amaral ◽  
Lisa Marshall ◽  
Shu-Gui Huang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
High Throughput ◽  
Mitochondrial Membrane ◽  
Yeast Cells ◽  
High Throughput Assay

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

2002 ◽  
Vol 7 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Shu-Gui Huang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mitochondrial Membrane ◽  
Living Cells ◽  
Screening Assay ◽  
Mitochondrial Inner Membrane ◽  
Obesity And Diabetes ◽  
High Throughput Screening Assay

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.

scholarly journals Proteasome impairment induces recovery of mitochondrial membrane potential and an alternative pathway of mitochondrial fusion

2015 ◽  
pp. MCB.00920-15 ◽  
Author(s):  
Ryohei Shirozu ◽  
Hideki Yashiroda ◽  
Shigeo Murata
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Alternative Pathway ◽  
Mitochondrial Fusion ◽  
Yeast Cells ◽  
Mitochondrial Quality Control ◽  
Independent Manner ◽  
Cellular Integrity

Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment.

scholarly journals T-2307 Causes Collapse of Mitochondrial Membrane Potential in Yeast

2012 ◽  
Vol 56 (11) ◽  
pp. 5892-5897 ◽  
Author(s):  
Tatsuya Shibata ◽  
Toshinari Takahashi ◽  
Eio Yamada ◽  
Akiko Kimura ◽  
Hiroshi Nishikawa ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Liver Mitochondria ◽  
Yeast Cells ◽  
Rat Liver Mitochondria ◽  
Content Type ◽  
Glycerol Medium

ABSTRACTT-2307, an arylamidine compound, has been previously reported to have broad-spectrumin vitroandin vivoantifungal activities against clinically significant pathogens, includingCandidaspecies,Cryptococcus neoformans, andAspergillusspecies, and is now undergoing clinical trials. Here we investigated the mechanism of action of T-2307 using yeast cells and mitochondria isolated from yeast and rat liver. Nonfermentative growth ofCandida albicansandSaccharomyces cerevisiaein glycerol medium, in which yeasts relied on mitochondrial respiratory function, was inhibited at 0.001 to 0.002 μg/ml (0.002 to 0.004 μM) of T-2307. However, fermentative growth in dextrose medium was not inhibited by T-2307. Microscopic examination using Mitotracker fluorescent dye, a cell-permeant mitochondrion-specific probe, demonstrated that T-2307 impaired the mitochondrial function ofC. albicansandS. cerevisiaeat concentrations near the MIC in glycerol medium. T-2307 collapsed the mitochondrial membrane potential in mitochondria isolated fromS. cerevisiaeat 20 μM. On the other hand, in isolated rat liver mitochondria, T-2307 did not have any effect on the mitochondrial membrane potential at 10 mM. Moreover, T-2307 had little inhibitory and stimulatory effect on mitochondrial respiration in rat liver mitochondria. In conclusion, T-2307 selectively disrupted yeast mitochondrial function, and it was also demonstrated that the fungal mitochondrion is an attractive antifungal target.

scholarly journals Aging Yeast Cells Undergo a Sharp Entry into Senescence Unrelated to the Loss of Mitochondrial Membrane Potential

Cell Reports ◽  
2013 ◽  
Vol 5 (6) ◽  
pp. 1589-1599 ◽  
Author(s):  
Steffen Fehrmann ◽  
Camille Paoletti ◽  
Youlian Goulev ◽  
Andrei Ungureanu ◽  
Hugo Aguilaniu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Yeast Cells

scholarly journals Application of a homogenous membrane potential assay to assess mitochondrial function

2012 ◽  
Vol 44 (9) ◽  
pp. 495-503 ◽  
Author(s):  
Srilatha Sakamuru ◽  
Xiao Li ◽  
Matias S. Attene-Ramos ◽  
Ruili Huang ◽  
Jianming Lu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
High Throughput ◽  
Mitochondrial Function ◽  
Hepg2 Cells ◽  
High Throughput Screening ◽  
Mitochondrial Membrane ◽  
Fluorescent Dyes ◽  
Screening Platform ◽  
Pharmacologically Active

Decreases in mitochondrial membrane potential (MMP) have been associated with mitochondrial dysfunction that could lead to cell death. The MMP is generated by an electrochemical gradient via the mitochondrial electron transport chain coupled to a series of redox reactions. Measuring the MMP in living cells is commonly used to assess the effect of chemicals on mitochondrial function; decreases in MMP can be detected using lipophilic cationic fluorescent dyes. To identify an optimal dye for use in a high-throughput screening (HTS) format, we compared the ability of mitochondrial membrane potential sensor (Mito-MPS), 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide, rhodamine 123, and tetramethylrhodamine to quantify a decrease in MMP in chemically exposed HepG2 cells cultured in 1,536-well plates. Under the conditions used, the optimal dye for this purpose is Mito-MPS. Next, we developed and optimized a homogenous cell-based Mito-MPS assay for use in 1,536-well plate format and demonstrated the utility of this assay by screening 1,280 compounds in the library of pharmacologically active compounds in HepG2 cells using a quantitative high-throughput screening platform. From the screening, we identified 14 compounds that disrupted the MMP, with half-maximal potencies ranging from 0.15 to 18 μM; among these, compound clusters that contained tyrphostin and 3′-substituted indolone analogs exhibited a structure-activity relationship. Our results demonstrate that this homogenous cell-based Mito-MPS assay can be used to evaluate the ability of large numbers of chemicals to decrease mitochondrial function.

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