Dana Gášková

Effect of high-voltage electric pulses on yeast cells: Factors influencing the killing efficiency

Abstract The decisive factors determining the killing efficiency of single rectangular electric pulses of 4–28 kV cm−1 amplitude and 1–300 μs duration in Saccharomyces cerevisiae S6/1 are pulse amplitude and duration, cell size and growth phase, post-pulse temperature and medium conductivity. In S. cerevisiae, the minimum pulse duration ensuring substantial killing is about 10 μs, the minimum amplitude being about 2 kV cm−1. The critical pulse-induced transmembrane breakthrough voltage is 0.75 V. A pulse-induced increase in membrane permeability for small species such as inorganic ions suffices to cause cell death. A preset killing rate can be achieved by varying pulse amplitude inversely to pulse duration. Comparison of killing data on S. cerevisiae S6/1 with those on the smaller-celled Kluyveramyces lactis showed the killing pulse amplitude to be roughly proportional to cell size except for low pulse amplitudes, at which smaller cells are much more killing-prone. In exponential S. cerevisiae cells increased pulse amplitude caused a sharp increase in killing while in stationary cells this effect was much lower and occurred only at pulse amplitude above 15–20 kV cm−1. Elevated post-pulse temperature lowered the killing rate whereas lowered temperature promoted it, probably by affecting the pore resealing. Lowering medium conductivity from 66 to 46 μS m−1 by suspension washing reduced the killing rate by 6–20%. Reproducible killing or electroporation therefore requires standardized cell concentration, and number of cell washings.

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.

Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay

No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.

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.

Monitoring the kinetics and performance of yeast membrane ABC transporters by diS-C3(3) fluorescence.

Kinetic features (initial start-up phase, drug pumping velocity and efficiency as dependent on drug concentration and growth phase) of yeast plasma membrane multidrug resistance ABC pumps were studied by monitoring the uptake of the fluorescent potentiometric dye diS-C3(3), which has been found to be expelled from the cells by these pumps. The monitoring was done with Saccharomyces cerevisiae mutants AD1-8 and AD1-3 deleted in different ABC pumps, and in their pump-competent parent strain US50-18C overexpressing transcriptional activators Pdr1p and Pdr3p. On addition to the cells, diS-C3(3) is expelled by the Pdr5p, Yor1p and Snq2p pumps with overlapping substrate specificity. The pump action can be assessed as a difference between the dye uptake curve for pump-competent and pump-deleted cells. The pump-mediated dye efflux, which shows an initial lag of various lengths, maintains a certain residual intracellular dye level. In the absence of external glucose the dye efflux ability of the pumps depends on the growth phase; late exponential and stationary cells can maintain the export for tens of minutes, whereas exponential cells keep up the pump action for limited time periods. This may reflect an insufficient number of pump molecules in the membrane or an effect of insufficient pump energization from endogenous sources. This effect is not mediated by changes in membrane potential because lowered membrane potential caused by inhibition of the plasma membrane H+-ATPase does not affect the pump action.

Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p

We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C(3)(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C(3)(3) by both pumps and confirmed the activation of membrane H(+)-ATPase by CCCP.

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast

The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max). These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.

Monitoring of real changes of plasma membrane potential by diS-C 3 (3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3′-dipropylthiadicarbocyanine, diS-C3(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-C3(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-C3(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.

Synchronous plasma membrane electrochemical potential oscillations during yeast colony development and aging

Microorganisms that survive in natural environments form organized multicellular communities, biofilms and colonies with specific properties. During stress and nutrient limitation, slow growing and senescent cells in such communities retain vital processes by maintaining plasma membrane integrity and retaining the ability to generate transmembrane electrochemical gradients. We report the use of a Saccharomyces cerevisiae colonial model to show that population growth in a multicellular community depends on nutrient diffusion and that resting cells start to accumulate from the beginning of the second acidic phase of colony development. Despite differentiation of colony members, synchronous transmembrane potential oscillation was detected in the organized colony. The electrochemical membrane potential periodically oscillated at frequencies between those for circadian to infradian rhythms during colony aging and transiently decreased at time points previously linked with rebuilding of yeast metabolism. Despite extensive decreases in the intracellular ATP concentration and in the amount and activity of the plasma membrane proton pump during nutrient limited growth and colony aging, the transmembrane electrochemical potential appeared to be maintained above a level critical for population survival.

Chemosensitization of multidrug resistant Candida albicans by the oxathiolone fused chalcone derivatives

Three structurally related oxathiolone fused chalcone derivatives appeared effective chemosensitizers, able to restore in part sensitivity to fluconazole of multidrug-resistant C. albicans strains. Compound 21 effectively chemosensitized cells resistant due to the overexpression of the MDR1 gene, compound 6 reduced resistance of cells overexpressing the ABC-type drug transporters CDR1/CDR2 and derivative 18 partially reversed fluconazole resistance mediated by both types of yeast drug efflux pumps. The observed effect of sensitization of resistant strains of Candida albicans to fluconazole activity in the presence of active compounds most likely resulted from inhibition of the pump-mediated efflux, as was revealed by the results of studies involving the fluorescent probes, Nile Red, Rhodamine 6G and diS-C3(3).