Jaroslav Večeř

Fluorescent probing of membrane potential in walled cells: diS‐C3(3) assay in Saccharomyces cerevisiae

Membrane‐potential‐dependent accumulation of diS‐C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λmax, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential‐reporting parameter, the λmax shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential‐dependent red λmax shift depends on relative cell‐to‐probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×106 cells/ml) and probe (10−7 M) concentrations at which a clearly defined relationship exists between the λmax shift and the potential‐dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10–30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n=30) are stained much faster and the λmax is more red‐shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n=44). At a suitable cell and probe concentration and under standard conditions, the λmax shift of diS‐C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Monitoring of membrane potential changes inSaccharomyces cerevisiae by diS-C3(3) fluorescence

Attempt was made to measure the membrane potential in yeast cells by the electrochromic probe di-4-ANEPPS (dibutylaminonaphthylethylene pyridinium propyl sulfonate) which has previously been used for measuring action potentials in neurons [1, 2]. This probe is believed to provide fluorescent response to changes in transmembrane electric field in nanoseconds by changing its fluorescence intensity due to an underlying wavelength shift of emission maximum. The requirements for successful measurement are (1) defined dependence of the fluorescence response on change in membrane potential, (2) low probe toxicity at the concentrations used, (3) reproducible incorporation of the probe solely into the outer layer of the membrane lipid bilayer (incorporation into the inner layer would give rise to two probe pools whose respective responses to membrane potential changes would be mutually opposite, hampering the measurement), (4) absence of any penetration of the probe into the cell. The fluorescence of the electrochromic probe was measured in suspensions of intact cells, protoplasts and phosphatidylserine/phosphatidylcholine (20/80) liposomes. Tentative adjustment of membrane potential was done by incubating the samples in 3.5-150 mmol/L KC1, the overall molarity being adjusted in each case to 150 mmol/L by choline chloride. The effect of nonuniform staining of individual cells on the excitation spectrum of the probe was eliminated by measuring the ratio of fluorescence intensities at excitation wavelengths of 450 and 530 nm [3, 4]. The measurements showed that (1) the probe responds to membrane potential change by an electrochromic shift; (2) the cell wall hampers the penetration of the probe to the plasma membrane of yeast cells; (3) the actual equilibration of the probe in cell suspension should take 10-15 min but in fact the staining intensity keeps on rising even at longer intervals; (4) this is due to the fact that the probe is not incorporated solely into the plasma membrane but spreads gradually into the cells and liposomes, which causes persistent variations in fluorescence response to membrane potential change. This penetration brings about a fluorescence change mimicking a decrease in membrane potential, i.e. membrane depolarization. The probe is therefore suitable for monitoring membrane potential in yeast only over short periods of time (up to 30 min). Longer monitoring will require either a modified staining protocol or 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.

Fluorescence Behavior of the pH-Sensitive Probe Carboxy SNARF-1 in Suspension of Liposomes

Abstract When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5′(and 6′)-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1′(3H)-isobenzofuran]-3′-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.

Glycine-Rich Loop of Mitochondrial Processing Peptidase α-Subunit Is Responsible for Substrate Recognition by a Mechanism Analogous to Mitochondrial Receptor Tom20

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) alpha-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast alpha-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility. Moreover, time-resolved measurements showed that a functionless alpha-MPP with a single-residue deletion in the loop (Y303W/DeltaG292) is defective particularly in the primary contact with substrate. Thus, the GRL was proved to be part of a contact site of the enzyme specifically recognizing the substrate. Regarding the surface exposure and presence of the hydrophobic patches within the GRL, we proposed a functional analogy between the presequence recognition by the hydrophobic binding groove of the Tom20 mitochondrial import receptor and the GRL of the alpha-MPP. A molecular dynamics (MD) simulation of the MPP-substrate peptide complex model was employed to test this hypothesis. The initial positioning and conformation of the substrate peptide in the model fitting were chosen based on the analogy of its interaction with the Tom20 binding groove. MD simulation confirmed the stability of the proposed interaction and showed also a decrease in GRL flexibility in the presence of substrate, in agreement with fluorescence measurements. Moreover, conserved substrate hydrophobic residues in positions +1 and -4 to the cleavage site remain in close contact with the side chains of the GRL during the entire production part of MD simulation as stabilizing points of the hydrophobic interaction. We conclude that the GRL of the MPP alpha-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H+-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane. The generated Delta(psi) was stable at least over tens of minutes. An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

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 (ΔμH+) across the microbial or plant cell membrane. Δψ- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Δψ-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Δψ generated by the reconstituted H+-ATPase. Its Δψ 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, Δψ and ΔpH. 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.

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells

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.

Speed of Accumulation of the Membrane Potential Indicator diS-C3(3) in Yeast Cells

The carbocyanine dye diS-C3(3) (3,3’- dipropylthiacarbocyanine iodide), whose the steady-state fluorescence spectra were measured in yeast cell suspensions, belongs to the group of slow (Nernstian, or redistribution) dyes which report on membrane potential by their voltage-sensitive partition between the extracellular medium and the cytosol.1–3 Since the emission spectrum shifts and the quantum yield of fluorescence increases upon binding of the dye in the cell, two fluorescence parameters,4–5 the wavelength of emission maximum and the intensity of fluorescence at this wavelength, were used to monitor the redistribution of the dye inside/outside the cells. To demonstrate that the dye accumulation in cells, as revealed by observed fluorescence changes, is actually membrane-potential-driven we used the uncoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone) which drastically increases membrane permeability for protons and depolarizes the cell membrane.6

Study of membrane potential changes of yeast cells caused by killer toxin K1.

transport of acetic acid and other weak organic acids in the strain Zygosaccharomyces bailii IGC 1307 and its regulation by several carbon sources. When transport was measured in cells grown in a medium with glucose or fructose, with labelled acetic acid at concentrations from 0.1 to 12 mmol/L, pH 5.0, the Lineweaver-Burk plot of the initial uptake rates was linear and consistent with a Michaelis-Menten kinetics. Acetic acid uptake (pH 5.0) was accompanied by the disappearance of extracellular protons, the uptake rates of which also followed Michaelis-Menten kinetics as a function of the acid concentration. The results indicated a proton symport of the anion form of the acid. Transport of labelled acetic acid at pH 5.0 was accumulative, the accumulation ratio in terms of free acid being about 50. Furthermore, the accumulated acid flew out of the cells after the addition of cold acetic acid as well as of benzoic, sorbic or pentanoic acids. The results suggested that all these acids may use the same carrier. Accordingly, benzoic, sorbic or pentanoic acids were competitive inhibitors of the acetic acid transport at pH 5.0. As expected, their transport was also associated with proton uptake that followed Michaelis-Menten kinetics. Apparently, neither propionic acid nor lactic acid used this transport system since they were not competitive inhibitors of acetic acid transport. Furthermore, when either acid was added to a cell suspension no transient external alkalinization indicative of proton uptake was observed. Cells of Z. bailii grown in a medium with either acetic acid or ethanol as the carbon and energy source were also analyzed for their capacity to transport acetic acid and the other weak organic acids mentioned above. The data indicated that, under these growth conditions, a mediated transport system for acetic was present and probably an acetate proton symport was again involved. However, the carrier appeared to be less specific, being able to accept not only benzoic, sorbic and pentanoic acids but also propionic and formic acids.

Is a Potential-Sensitive Probe diS-C3(3) a Nernstian Dye?: Time-Resolved Fluorescence Study with Liposomes as a Model System

Fluorescence probe diS-C3(3) (3,3′-dipropylthiacarbocyanine iodide) is a redistribution dye used for the assessment of diffusion membrane potential in living cells. Despite of a wide usage of the dye, detailed mechanisms of its fluorescence response on changes of the membrane potential have not been well documented yet. Studies, where diS-C3(3) was used as an indicator of the membrane potential, involve an implicit assumption that diS-C3(3) is a ‘Nernstian dye’, eg. that the dye redistributes between the extracellular medium and cytoplasm according to a diffusion membrane potential and its equilibrium concentrations inside and outside the cell follow the Nernst equation. Validity of this assumption is critical when diS-C3(3) is used for a quantitative assessment of the membrane potential.