The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials physiology   MeSH
• Rhodotorula cytology   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.

• MeSH
• Arylsulfonates chemistry   MeSH
• Models, Biological * MeSH
• Cell Membrane chemistry   physiology   MeSH
• Electrochemistry MeSH
• Fluorescent Dyes analysis   MeSH
• Spectrometry, Fluorescence MeSH
• Isoxazoles chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials MeSH
• Proteolipids chemistry   MeSH
• Proton-Translocating ATPases chemistry   MeSH
• Protons * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arylsulfonates MeSH
• Fluorescent Dyes MeSH
• Isoxazoles MeSH
• oxonol V MeSH Browser
• Proteolipids MeSH
• proteoliposomes MeSH Browser
• Proton-Translocating ATPases MeSH
• Protons * MeSH
• pyranine MeSH Browser

Dysfunction of mitochondrial ATPase (F1F(o)-ATP synthase) due to missense mutations in ATP6 [mtDNA (mitochondrial DNA)-encoded subunit a] is a frequent cause of severe mitochondrial encephalomyopathies. We have investigated a rare mtDNA mutation, i.e. a 2 bp deletion of TA at positions 9205 and 9206 (9205DeltaTA), which affects the STOP codon of the ATP6 gene and the cleavage site between the RNAs for ATP6 and COX3 (cytochrome c oxidase 3). The mutation was present at increasing load in a three-generation family (in blood: 16%/82%/>98%). In the affected boy with severe encephalopathy, a homoplasmic mutation was present in blood, fibroblasts and muscle. The fibroblasts from the patient showed normal aurovertin-sensitive ATPase hydrolytic activity, a 70% decrease in ATP synthesis and an 85% decrease in COX activity. ADP-stimulated respiration and the ADP-induced decrease in the mitochondrial membrane potential at state 4 were decreased by 50%. The content of subunit a was decreased 10-fold compared with other ATPase subunits, and [35S]-methionine labelling showed a 9-fold decrease in subunit a biosynthesis. The content of COX subunits 1, 4 and 6c was decreased by 30-60%. Northern Blot and quantitative real-time reverse transcription-PCR analysis further demonstrated that the primary ATP6--COX3 transcript is cleaved to the ATP6 and COX3 mRNAs 2-3-fold less efficiently. Structural studies by Blue-Native and two-dimensional electrophoresis revealed an altered pattern of COX assembly and instability of the ATPase complex, which dissociated into subcomplexes. The results indicate that the 9205DeltaTA mutation prevents the synthesis of ATPase subunit a, and causes the formation of incomplete ATPase complexes that are capable of ATP hydrolysis but not ATP synthesis. The mutation also affects the biogenesis of COX, which is present in a decreased amount in cells from affected individuals.

• MeSH
• Electrophoresis, Gel, Two-Dimensional methods   MeSH
• Adenine metabolism   MeSH
• Adenosine Triphosphate biosynthesis   MeSH
• Adenosine Triphosphatases chemistry   physiology   MeSH
• Fibroblasts chemistry   enzymology   metabolism   pathology   MeSH
• Intracellular Membranes chemistry   enzymology   MeSH
• Cells, Cultured MeSH
• Skin pathology   MeSH
• Humans MeSH
• Membrane Potentials genetics   MeSH
• RNA, Messenger biosynthesis   MeSH
• DNA, Mitochondrial biosynthesis   genetics   MeSH
• Mitochondrial Proton-Translocating ATPases biosynthesis   MeSH
• Mitochondria chemistry   enzymology   MeSH
• Mutation genetics   MeSH
• Child, Preschool MeSH
• Electron Transport Complex IV biosynthesis   chemistry   metabolism   physiology   MeSH
• Sequence Deletion genetics   MeSH
• Oxygen Consumption genetics   physiology   MeSH
• Thymidine metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Adenosine Triphosphate MeSH
• Adenosine Triphosphatases MeSH
• RNA, Messenger MeSH
• DNA, Mitochondrial MeSH
• Mitochondrial Proton-Translocating ATPases MeSH
• MT-ATP6 protein, human MeSH Browser
• Electron Transport Complex IV MeSH
• Thymidine MeSH

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.

• MeSH
• Fluorescent Dyes metabolism   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines metabolism   MeSH
• Membrane Potentials physiology   MeSH
• Saccharomyces cerevisiae physiology   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

• MeSH
• Staining and Labeling MeSH
• Cell Membrane metabolism   physiology   MeSH
• Cell Wall metabolism   MeSH
• Time Factors MeSH
• Fluorescence MeSH
• Microscopy, Confocal MeSH
• Liposomes metabolism   MeSH
• Membrane Potentials * MeSH
• Pyridinium Compounds metabolism   MeSH
• Saccharomyces cerevisiae metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine MeSH Browser
• 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Browser
• Liposomes MeSH
• Pyridinium Compounds MeSH

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

• MeSH
• Artifacts MeSH
• Benzothiazoles MeSH
• Time Factors MeSH
• Fluorescence MeSH
• Fluorescent Dyes * MeSH
• Fluorometry * MeSH
• Indicators and Reagents MeSH
• Carbocyanines * MeSH
• Membrane Potentials * MeSH
• Organometallic Compounds MeSH
• Flow Cytometry methods   MeSH
• Pyridinium Compounds analysis   metabolism   MeSH
• Scattering, Radiation MeSH
• Saccharomyces cerevisiae physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Browser
• 3,3'-dipropyl-2,2'-thiadicarbocyanine MeSH Browser
• 3,3'-dipropyloxadicarbocyanine MeSH Browser
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Benzothiazoles MeSH
• Fluorescent Dyes * MeSH
• Indicators and Reagents MeSH
• Carbocyanines * MeSH
• Organometallic Compounds MeSH
• Pyridinium Compounds MeSH
• tetramethyl rhodamine ethyl ester MeSH Browser