Jaromír Plášek

SLOW FLUORESCENT INDICATORS OF MEMBRANE POTENTIAL : A SURVEY OF DIFFERENT APPROACHES TO PROBE RESPONSE ANALYSIS

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.

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.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Time-resolved polarized fluorescence studies of the temperature adaptation in Bacillus subtilis using DPH and TMA-DPH fluorescent probes

The validity of the concept of homeoviscous adaptation was tested for bacteria Bacillus subtilis. The Bacillus subtilis grown at 20 degrees C (referred to as Bs20) exhibit a considerable increase of branched anteiso-C15, the major fatty acid component of membrane lipids, relative to membranes grown at 40 degrees C (Bs40). The time-resolved fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) showed that these changes in the lipid composition are accompanied by changes in a mean lipid order. In particular, the DPH order parameters and measured in Bs20 membranes at 18 degrees C and in Bs40 membranes at 45 degrees C, respectively, tend to be equal. This effect was less pronounced for TMA-DPH. Our observations suggest that a physical parallel to the changes of lipid composition is the maintenance of an optimal lipid order in the hydrophobic core of the cytoplasmic membranes. It can be interpreted as a tendency of Bacillus subtilis to keep the lateral pressure in its membranes at an optimal value, independent of the temperature of cultivation.

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.

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.

Phe475 and Glu446 but not Ser445 participate in ATP-binding to the α-subunit of Na+/K+-ATPase

Abstract The ATP-binding site of Na + /K + -ATPase is localized on the large cytoplasmic loop of the α-subunit between transmembrane helices H 4 and H 5 . Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser 445 , Glu 446 , and Phe 475 were proposed to be close to the binding pocket. Replacement of Phe 475 with Trp and Glu 446 with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser 445 with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser 445 is close to the binding site, although it does not participate in binding.

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.

Transmembrane potential measurement with carbocyanine dye diS-C3-(5): fast fluorescence decay studies.

The mechanism of carbocyanine dye diS-C3-(5) fluorescence intensity variations with transmembrane potential changes has been studied using time-resolved fluorescence spectroscopy. Clear evidence is given of the transmembrane-potential-dependent partition of the dye among various sites with different fluorescence lifetimes. It was found that fluorescence decay profiles reflect the transmembrane potential changes.