Enhanced Sensitivity of pHluorin-Based Monitoring of Intracellular pH

Technical Note pubs.acs.org/ac

Enhanced Sensitivity of pHluorin-Based Monitoring of Intracellular pH Changes Achieved through Synchronously Scanned Fluorescence Spectra Jaromír Plásě k,* Adéla Melcrová, and Dana Gásǩ ová

Downloaded by FLORIDA ATLANTIC UNIV on September 13, 2015 | http://pubs.acs.org Publication Date (Web): September 9, 2015 | doi: 10.1021/acs.analchem.5b02779

Charles University, Faculty of Mathematics and Physics, Ke Karlovu 3, 12116 Prague, Czech Republic ABSTRACT: Since its introduction in 1998, genetically encoded pH-sensitive sensor ratiometric pHluorin proved to be a valuable tool for cell physiology studies. Here, we show how the sensitivity of pHluorin-based monitoring of intracellular pH changes performed with cell suspensions can be enhanced by using synchronously scanned fluorescence spectroscopy. In the suspensions of S. cerevisiae cells subjected to varying extracellular pH values, we have been able to measure statistically significant changes in intracellular pH of less than 0.1 unit, which were not detectable using a standard ratiometric approach.

fluorescent probe assays in their intracellular pH and other physiological parameters are often performed using cells suspensions in cuvettes. As shown in the present paper, when cell suspensions are studied, correction for autofluorescence background can be considerably simplified and the sensitivity of pH assays enhanced if synchronously scanned florescence spectra of pHluorin are measured instead of the plain excitation spectra. Synchronously scanned fluorescence spectroscopy (SSF) involves the registration of the fluorescence intensity while simultaneously scanning both fluorescence excitation and emission wavelengths, keeping the wavelength difference (offset) between them constant.11−13 The main advantage of the SSF spectra compared to the common fluorescence excitation and emission spectra consists of their narrow and symmetrical shapes, which facilitates discrimination between multiple spectral components of heterogeneous fluorescence.

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early all reactions in living cells are pH dependent, making the tight regulation of intracellular pH (pHi) a crucial task of any living cell.1−4 Dramatic differences in cell behavior may stem from a relatively small decrease in the intracellular pH of about 0.3−0.4 pH units. Therefore, accurate and feasible tools to monitor intracellular pHi in living cells are required to advance further understanding of cellular functioning. Current techniques used to measure pH were briefly reviewed by Orij et al.5 An important step in measuring the intracellular pH, including a selective measurement of specific pH in cellular organelles, has been accomplished by the development of genetically encoded pH-sensitive sensors derived from green fluorescent protein (GFP). More information on the GFP-based pH indicators for in vivo use can be found in several recent reviews.1,6,7 Ratiometric pHluorin, one of the most successful members of this family, was prepared by Miesenbock et al. via a systematic histidinebased combinatorial mutagenesis of seven key residues in the vicinity of GFP chromophore.8 This variant of GFP displays an increase in fluorescence excited around 475 nm concomitantly with a decrease in fluorescence excited around 395 nm upon a pH shift from 7.5 to 5.5 (with apparent pKa of 6.9). The photophysical properties of ratiometric pHluorin, including the relationship between its protonated and deprotonated states and fluorescence spectra, seem to be identical with the properties of GFP itself; for which see, e.g., review by Tsien.9 A notorious problem of many fluorescent probe assays is the existence of cell autofluorescence background that is mixed with the probe signal. A normal correction for the interfering background consists of measuring the autofluorescence in control samples of untransformed, pHluorin-free cells, which is then subtracted from the complex fluorescence of cells with pHluorin.10 As far as the yeast and bacteria are concerned, © XXXX American Chemical Society

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MATERIALS AND METHODS

Yeast Strains. We used S. cerevisiae strains derived from BY4741 (MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; EUROSCARF), a mutant expressing ratiometric pHluorin in its cytosol and a control strain containing identical plasmid without the pHluorin gene. These strains were kindly provided by Dr. Hana Sychrova, Institute of Physiology, Czech Academy of Sciences, Prague. For details about the construction of these strains, see Maresova et al.14 Yeast Growth and Sample Preparation. Cells were grown in synthetic yeast nitrogen base medium (0.67% YNB Received: July 23, 2015 Accepted: September 1, 2015

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DOI: 10.1021/acs.analchem.5b02779 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note


Analytical Chemistry

and 470 nm (integrated over intervals 408−412 and 468−472 nm, respectively). Their ratio, hereafter referred to as R410/470, was used for further evaluation of pH-dependent changes in the excitation spectra of pHluorin fluorescence. In the case of SSF spectra, the autofluorescence measured in control samples was first scaled to the intensity of autofluorescence observed in the spectra of pHluorin-labeled cells, as described under Results and Discussion. Then, this scaled autofluorescence was subtracted from the particular raw SSF spectrum, and finally, a R110/30 ratio has been assessed, which is based on fluorescence intensity values measured in the maxima of the SSF peaks: for the offset of 110 nm at λexc = 394 nm and for 30 nm offset at λexc = 473 nm (integrated over intervals of 392−396 and 470−475 nm, respectively). Calibration of pH Measurements. Ratiometric pHluorin was used to determine intracellular pH and its changes. For the determination of the relationship between pHluorine fluorescence spectra and pHi, we used the method published by Maresova et al.,14 which has been derived from Brett et al.:10 Calibration buffers contained 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 200 mM ammonium acetate, 10 mM NaN3, and 10 mM 2-deoxyglucose. Their pH values were adjusted with NaOH or HCl. To obtain the calibration curve, the cells were resuspended in a series of calibration buffers with pH from 5.5 to 7.6. This range is adequate for the yeast cell assays and matches the calibration protocol used by Miesenbock et al.8 Calibration curves presented in Figure 3 are based on data from three experiments (three different cultures used to prepare three sets of cell suspensions, each of different cell concentration, −1.7, 5.1, and 8.5 × 106 cells/mL). The calibration curves were constructed for both synchronously scanned fluorescence spectra and standard excitation spectra. The excitation spectra were measured with λem = 520 nm to match the settings used in preceding studies by Maresova et al.14 and Valkonen et al.15 For measuring intracellular pHi, washed intact cells were resuspended in MES-TEA buffers of defined pH and composition, keeping their concentrations within the above range.

without amino acids, 2% glucose, auxotrophic supplements). Brent supplement mixture of amino acids (BSM-ura) was added to the final concentration of 1.5 mg/mL. The cells were harvested in postdiauxic phase of growth (18−22 h after inoculation to a fresh YNB medium), washed twice with MESTEA buffer (25 mM MES hydrate, pH adjusted by TEA to 6.8), and finally suspended to a desired concentration in MES-TEA buffers of defined pH. The cell concentration was set using cell suspension optical density OD578 (measured at 578 nm using Novaspec III spectrophotometer, Amersham Biosciences). Corresponding cell counts were measured with a Cellometer Vision CBA image cytometer (Nexcelom Biosciences). Fluorescence Spectroscopy. Fluorescence spectra of cell suspensions were measured in disposable 1 × 1 cm UV-grade fluorometric cuvettes (Kartell, Italy) using Fluoromax-3 spectrofluorometer (Jobin-Yvon Horiba). Standard spectroscopy was performed using slit width set to Δλexc = 1 nm and Δλem = 10 nm (excitation spectra) and Δλexc = 10 nm and Δλem = 1 nm (emission spectra). For each sample, two SSF spectra were measured using offsets of 30 and 110 nm (both slit widths set to 3 nm). In most cases, the OD578 of cell suspensions was set to 0.4 (≈2.7 × 106 cells/mL). The relative intensities of all ten spectra in Figure 1 have been scaled to match actual

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Figure 1. Fluorescence spectral properties of ratiometric pHluorin expressed in the cytosol of S. cerevisiae yeast as measured with permeabilized cells suspended in buffers of pH = 5.3 (black lines) and pH = 7.6 (gray lines). Thick full lines represent excitation measured with λem = 508 and emission spectra measured with λexc = 450 and 385 nm for pH = 5.3 and 7.6, respectively, as obtained after the subtraction of autofluorescence (thin full lines; measured in untransformed control cells) from raw spectra. To avoid an unclear tangle of curves in the emission part of this figure, raw excitation spectra only are shown here (dashed lines). Relative intensities of all ten spectra match the fluorescence intensities measured with instrument setting described under the Materials and Methods. Arrows indicate the size of an optimal synchroscan offset.

RESULTS AND DISCUSSION Synchronously Scanned Fluorescence Spectroscopy Makes It Possible to Discriminate between Fluorescence of Ratiometric pHluorin and Autofluorescence of the Cells. In order to fully exploit the advantages of the SSF spectroscopy, the selection of offset values must be consistent with the spectral properties of examined fluorochromes. Therefore, we first checked the actual parameters of pHluorin fluorescence excitation and emission spectra in our cell samples and compared them with the related autofluorescence spectra from untransformed control cells. Permeabilized cells were used for this purpose to get defined pH values (5.5 and 7.6) of the pHluorin environment in the cell cytosol. The spectra shown in Figure 1 match all known key features of pHluorin fluorescence, as presented in the existing literature on this subject. In particular, the excitation spectra exhibit two well-separated bands whose relative intensities vary with ambient pH. Moreover, the spectral maximum of the right excitation band shifts about 6 nm toward longer wavelengths with the ambient pH increased from 5.5 to 7.6. The emission spectra, which were measured with λexc = 450 and 385 nm for pH = 5.5 and 7.6, respectively, depend only little on both pH and λexc, apart from a spectral shift of about 6 nm, which is similar to the above-mentioned shift observed in the right

fluorescence intensities measured with instrument setting presented above. Both the fluorescence excitation spectra and SSF spectra were corrected for the spectral variations of the excitation light intensity monitored by the photodiode reference detector of the spectrofluorometer. For each pHluorin-labeled cell suspension, excitation and SSF spectra of autofluorescence from its pHluorin-free control were also acquired. The autofluorescence excitation spectra were directly subtracted from the raw fluorescence excitation spectra of pHluorin-labeled cells. Finally, relative fluorescence intensities were read from the excitation spectra for λexc = 410 B


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fluorescence. Figure 2B illustrates how the corrected SSF peaks vary with the ambient pH of pHluorin. Note that for the peak of 110 nm offset a visible long-wavelength shoulder occurs alongside its red edge if pH drops below 6. The SSF Calibration Curve for the Conversion of pHluorin Fluorescence Spectra to pH Values Is Steeper than the Common Ratiometric Curve. Calibration curves for the conversion of pHluorin fluorescence spectra to pH values were constructed for both the standard excitation spectra and synchronously scanned fluorescence, as described in Materials and Methods. The R410/470 and R110/30 ratios shown in Figure 3 were determined in three independent experiments.


excitation band. A typical contribution of the cell autofluorescence to complex raw fluorescence spectra of pHluorincontaining mutant yeast is illustrated in Figure 1. The sharpest synchronously scanned fluorescence spectra can be achieved when the offset is equal to the Stokes shift between the excitation and emission spectral maxima (a consequence of multiplication of two synchronously scanned spectral profiles). Given the excitation and emission spectra shown in Figure 1, we fixed the synchroscan offset to 30 and 110 nm for the long- and short-wavelength excitation band of pHluorin fluorescence, respectively. Resultant synchronously scanned fluorescence spectra of permeabilized yeast cells exhibit sharp pHluorin peaks that can be clearly discerned from the spectra of autofluorescence, which are seen to the left of the pHluorin peaks, Figure 2. Despite the fact that identical

Figure 3. Calibration curves of the pH-sensitive response of pHluorin fluorescence: black circles, synchronously scanned fluorescence (R110/30 ratios) of cell suspensions; unfilled triangles, synchronously scanned fluorescence of cell lysate; gray diamonds, standard excitation spectra (R410/470 ratios). Error bars indicate standard errors of the means from three independent experiments.

Very small standard errors of the means indicate high reproducibility of the calibration data. Upon a conversion of the curves in Figure 3 to pH versus ratio plots, these plots were successfully fitted by nonlinear regression (R = 0.9993) to following cubic polynomials, which has been used later as transform equations in the analysis of the next experimental data: pH = 4.3791 + 2.9025R110/30 − 0.9589(R110/30)2 + 0.1413(R110/30)3

Figure 2. Synchronously scanned fluorescence spectra of ratiometric pHluorin (gray lines, offset 110 nm; black lines, offset 30 nm). (A) Correction of raw SSF spectra for the autofluorescence. Thick dashed lines, raw spectra of permeabilized cells with pHluorin, pH = 7.0; thin dashed lines, autofluorescence from untransformed control cells; thin full lines, the same autofluorescence scaled to match the intensity of autofluorescence exhibited by the cells with pHluorin; thick full lines, autofluorescence-free spectra of pHluorin fluorescence. (B) The pH dependence of SSF spectra of ratiometric pHluorin: thick lines, pH = 7.6; thin lines, pH = 5.3.

(1a) 2

pH = 3.2171 + 6.5288R 410/470 − 3.5353(R 410/470) + 0.7378(R 410/470)3

(1b)

The respective calibration curves obtained for the SSF spectra and the standard excitation spectra exhibit very similar profiles, but the ΔR110/30/ΔpH slopes in the SSF curve are considerably steeper compared to the ΔR410/470/ΔpH slopes of the ratiometric curve (by a factor of about 1.5). This difference can be plausibly attributed to a multiplicative combination of the pH-dependent changes of the excitation and emission spectral bands in the SSF spectra of pHluorin. The SSF calibration curve was also compared with SSF data from cell lysate. Interestingly, the data from the whole cells and the cell lysate overlap perfectly for pH higher than 6.5, which is sufficient for the expected usual values of cytosolic pH in S. cerevisiae cells. Still, even when the calibration pH is close to 5.5, the above-mentioned difference is less than 0.2 pH units. An explanation of this difference poses obviously serious challenges in regard to the methods of intracellular pH

OD values were carefully set in samples with cells containing pHluorin and untransformed control cells, actual autofluorescence intensities measured in the respective samples have often been slightly different (up to about 10%). If this happens, the intensity of autofluorescence from the control cell sample can be scaled to match the spectral intensity within the discernible autofluorescence part of the raw SSF spectrum of the cells with pHluorin, Figure 2A. Such a scaled autofluorescence spectrum can then be subtracted from the corresponding raw spectrum to get a pure SSF spectrum of pHluorin C


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expression. When the autofluorescence contribution is less than 10% of the total signal, this correction ceases to be critical. The test for the ability of yeast cells to maintain stable intracellular pH was carried out with a series of cell suspensions in MES-TEA buffers of pH from 5.0 up to 7.8. The ratiometric analysis of standard excitation spectra performed in two independent experiments supports the expected independence of intracellular pH on extracellular pH, Figure 4. In contrast, the SSF analysis revealed a small, but statistically significant drop of intracellular pH occurring when the extracellular pH decreased from pH = 6.2 to pH = 5.5 (mean drop of 0.12 pH units; t-test with P-value

Enhanced Sensitivity of pHluorin-Based Monitoring of Intracellular pH

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