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1.Introductionis the most abundant intracellular divalent cation, which has a central role in the plant’s light-absorbing molecule chlorophyll, and is an essential cofactor of adenosine triphosphate (ATP), the “energy currency” of the cell. plays an important role in the regulation of more than 350 enzymes,1 particularly those dealing with DNA replication, transcription, and translation. has been proposed as a modulator of signaling2 and a direct second-messenger role was recently reported for this ion during the activation of T-lymphocytes after antigen receptor stimulation.3 Total concentration in mammalian cells is in the 17 to 20 mM range,4 however, most of it is considered to be bound to ATP, membranes, nucleic acids, proteins, or sequestered in intracellular organelles. Free concentration in cells is reported to be tightly regulated in the 0.25 to 1.5 mM range,5 however, under hormonal or chemical stimulation, large fluxes are observed which do not drastically alter the cytosolic free level.6 Several techniques, such as NMR, ion selective microelectrodes, and fluorescent -sensitive dyes, have been used to obtain information about free dynamics,7 but only fluorescent dyes allow visualization of intracellular at the single cell level with relatively high spatial resolution. A great disadvantage of these dyes is, however, in most cases, their intensiometric nature meaning that measurements are affected by cell movement, focal plane shift, bleaching, or dye local concentration change, impeding prolonged monitoring of cellular . Few ratiometric -sensitive dyes exist (for example mag-fura-2),8 but they require UV excitation which is harmful to the cell and requires a specialized optical setup.7 Most -sensitive dyes also have high sensitivity for (with affinity constants for in the micromolar range), so they are predominantly used as low affinity indicators.7 An exception is a recently developed class of indicators based on beta diketones (the KMG series), which display a high -over- selectivity [KMG-104 , ].9 An inconvenience of this group of dyes is, however, that they are also intensiometric thus hindering their use for prolonged imaging of intracellular . Recently, the first family of genetically encoded indicators for , MagFRET, was reported. MagFRETs are based on the human centrin HsCen3 moiety flanked by the cyan fluorescent protein (FP), Cerulean, and the yellow FP, Citrine, as a Förster resonance energy transfer (FRET) donor and acceptor, respectively.10 MagFRET variants showed affinity constants for in the 0.15 to 15 mM range, but the constructions with close to the physiological range (MagFRET1, 2, 7, 8) also displayed a relatively high affinity for in the 10 to range), thus potentially requiring attention when interference is possible. Expression of MagFRET1 in HEK293 cells without targeting sequences resulted, as in the case of -sensitive dyes, in cytosolic staining with a nonlabeled nucleus. But perhaps the main uncertainty regarding these indicators’ functionality comes from the apparent insensitivity to several stimuli previously reported to increase free cytosolic 10 although, on the other hand, -dependent ratio changes were further confirmed when cells were permeabilized and high concentrations of or ethylenediaminetetraacetic acid (EDTA) were applied. In our attempt, we developed an indicator for based on another non-FRET reporting mechanism. For our indicator, we used a novel FP functionalized to sense in the millimolar range. By addition of a second reference, FP, we conferred ratiometric properties to the indicator, thus making it insensitive to local variations in the fluorescence intensity caused by cell movement or defocusing. Different from other available indicators to date, our sensor, which we have called MagIC (indicator for Magnesium Imaging in Cell) localizes both in the cell nucleus and in the cytosol, allowing simultaneous imaging of in these compartments. In the present work, we have characterized this novel sensor and employed it for monitoring cytosolic under different conditions. 2.Materials and Methods2.1.Plasmid Construction, Protein Expression, and PurificationThree residues in circularly permuted (cp) 173Venus were replaced using the approach previously described by Sawano and Miyawaki,11 to obtain (S220E, S31D, Q33E)-cp173Venus. The sequence encoding this Venus variant was amplified using primers containing XhoI (forward) and EcoRI (reverse) restriction sites, and the open reading frame (ORF) was cloned into vector (Invitrogen, Carlsbad, California). ORFs of reference FPs were amplified using primers containing BamHI (forward) and XhoI (reverse) restriction sites and ligated in the vector containing the (S220E, S31D, Q33E)-cp173Venus between the XhoI restriction site and an upstream BamHI restriction site [Fig. 1(b)]. In the final indicator (see below), spontaneous mutations—G4S [enhanced green fluorescent protein (EGFP) 177S], V89I (EGFP 18V), and Q142M (EGFP 72Q)—were found in the -sensitive Venus moiety, so additional indicator variants were generated: (1) with reversed spontaneous mutations, (2) a variant holding all the acidic residues present in CatchER (F52E, T54E in the -sensitive Venus, in addition to those shown in Table 1),12 and (3) the reversed mutant holding only the five negatively charged residues of CatchER in the original cp173Venus moiety. All these mutations were introduced following the approach proposed by Sawano and Miyawaki11 and the subsequent construction process was identical to that described above. Table 1Amino acid replacements in the cp173Venus variant used in MagIC and corresponding mutations in EGFP-based CatchER.12
Mg2+/Ca2+-sensitive cp173Venus also contains the following mutations relative to the original cp173Venus: V89I, Q142M (in cp173Venus numeration). Protein expression was carried out using the JM109 (DE3) Escherichia coli strain. Culturing was performed in lysogeny broth medium at 23°C for 60 h with continuous shaking at 150 rpm. The bacterial pellet obtained after centrifugation was resuspended in phosphate-buffered saline (PBS) and mechanically lysed. The cleared lysate was purified using a Ni-NTA column (Qiagen, Hilden, Germany) and then gel-filtered in a PD-10 column (GE-Healthcare, Uppsala, Sweden) for replacement of the buffer with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4. For expression in mammalian cells, the indicator’s ORF was subcloned into the pcDNA3.2 vector (Life Technologies). For organelle targeting, subcloning was performed into modified pcDNA3.2 vectors containing either the human ornithine transcarbamylase signal sequence (for mitochondrial targeting)13 or the calreticulin signal sequence/KDEL motif [for endoplasmic reticulum (ER) targeting]. 2.2.Seminative Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Purified Protein Samples or HeLa Cells LysateThe purpose of seminative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was to conduct the separation of the proteins contained in the sample, preserving, at the same time, the ability of FPs to emit light when excited. For this purpose, the procedure for SDS PAGE did not include the step of heat treatment of the sample to avoid the denaturation of all contained proteins. Seminative SDS PAGE was carried either for the purified protein sample of MagIC or for the lysate of HeLa cells expressing MagIC in the cytosol. The protein sample was prepared by dilution with sample buffer containing 2% SDS, 62.5 mM Tris–HCl (pH 6.8), 5% (v/v) -mercaptoethanol, 10% glycerol (v/v) and bromophenol blue. The lysate of HeLa expressing cytosolic MagIC was prepared as follows: cells grown on a 10-cm plastic plate 24 h after transfection (see Sec. 2.5) were washed with 4 mL ice cold PBS, and then detached by scrapping and resuspended in 2 mL ice-cold PBS for washing. After a centrifugation step (, 10 min), the supernatant was removed and cells were resuspended in of the sample buffer described above and applied directly to the polyacrylamide gel. The separation (a variant of this procedure is provided in Ref. 14) was performed using 12% separating polyacrylamide gel in the presence of 10% SDS. 2.3.Spectral Characterization of the Mg2+/Ca2+-Sensitive Venus Fusion Constructions with Reference FPsFor the indicator variants (containing different reference FPs), characterization of the -sensitivity of the absorption, excitation, and emission spectra was conducted. For this, protein samples diluted in 20 mM HEPES-KOH buffer (pH 7.2) were analyzed in a V-630 Bio spectrophotometer (JASCO; for absorption measurements), in a F-2500 fluorescence spectrophotometer (Hitachi, Japan; for excitation spectra acquisition) or in F7000 fluorescence spectrophotometer (Hitachi; for emission spectra acquisition) either in the absence or presence of 5 mM . Absorption spectra were acquired in the 450 to 600 nm range. Excitation spectra for each of the constructions were acquired separately for the Venus variant (monitored wavelength corresponding to its emission maximum, 530 nm) and the corresponding reference FP (monitored at the respective emission maximum of the reference FP). These monitored wavelengths were, specifically, 610 nm for mCherry, 633 nm for mKate2, 563 nm for mKOκ, and 565 nm for the nonphotoconverted PSmOrange. After these separate measurements (for Venus and the reference FP) were conducted in the absence of , 5 mM were added into the same cuvette and the measurements were repeated. Similarly, emission measurements of the PSmOrange-[-sensitive cp173Venus], mKOκ-[-sensitive cp173Venus] and mKate2-[-sensitive cp173Venus] constructions were conducted. In this case, the Venus variant fluorescence and the emission of the reference FP were acquired using separate excitation. In all constructions, the -sensitive Venus was excited with 516-nm light (bandwidth 5 nm). Emissions of the corresponding reference FPs were obtained by excitation with the following wavelengths: 548 nm for PSmOrange, 551 nm for mKOκ, and 588 nm for mKate2. In detail, characterization of the -sensitivity of the emission spectrum of mCherry-[-sensitive cp173Venus] construction (MagIC) is provided in Sec. 2.4. 2.4.In Vitro Analysis of MagICAffinity and selectivity were determined by separate excitation of the -sensitive Venus variant (excitation 516 nm) and mCherry (excitation 587 nm) in an F-7000 fluorescence spectrophotometer (Hitachi). The indicator affinity was determined using serial dilutions of in a background of 100 mM KCl and 10 mM MOPS (pH 7.4, KOH). Selectivity (as the ability to displace at physiological concentrations from the created binding site) was assayed in solutions with 100 mM KCl and 1 mM , 10 mM MOPS (50 mM for the case of polyamines) (pH 7.4) and the ion of interest and then compared with a similar solution without that ion. pH sensitivity of the indicator was analyzed in solutions containing either 10 mM MES–HEPES (pH range 5.5 to 8.0) or Tris (pH 8.3 to 9.0) in a background of 100 mM KCl and 1 mM . 2.5.Cell Culture and TransfectionHeLa cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest) in a 5% atmosphere. Pituitary cells of the GH3 line (ATCC CCL-82.1) were maintained in DMEM/F12 + GlutaMAX (Gibco, Life Technologies) supplemented with 2.5% FBS (Biowest) and 15% heat inactivated horse serum (Gibco, Life Technologies) in 5% atmosphere. Cell transfection was carried as in the “-phosphate” method15 or with Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Simultaneous expression of different indicators was achieved by cotransfection with a mixture of plasmids containing the indicators. 2.6.General Imaging ConditionsCell imaging was performed 24 to 48 h after transfection using a Nikon Ti Eclipse confocal microscope equipped with oil immersion lens (NA: 1.4) and a Nikon A1 imaging system. Experiments, if not otherwise specified, were conducted at room temperature (around 25°C). 2.7.Imaging with Indicators in CellsIn the present work, three genetically encoded indicators were used: MagIC (or the negative control mCherry-cp173Venus), B-GECO1—a genetically encoded indicator [],16 and pHluorin—a genetically encoded pH indicator ( around 7.0).17 In experiments with cells expressing MagIC or the negative control mCherry-cp173Venus, the Venus variants were excited with a 488-nm laser and their emissions were collected in the 500 to 550 nm range. mCherry excitation was performed with a 561 nm laser and its emission was collected in the 570 to 620 nm range. When MagIC was coexpressed with B-GECO1,16 a genetically encoded indicator with excitation and emission maxima at 378 and 446 nm, respectively, this last was excited with a 403.5-nm laser, and emission was read in the 425 to 475 nm range. Channels were acquired sequentially. Excitation of ratiometric pHluorin was performed with 403.5 and 488 nm lasers and the corresponding emissions were read in the 500 to 550 nm range. 2.8.Image AnalysisImage analysis was performed with ImageJ and Metamorph (Molecular Devices, United States). MagIC ratio images were obtained dividing the Venus channel by the mCherry one after background subtraction. 2.9.In Situ Mg2+ Sensitivitysensitivity of the indicator expressed in cells was assayed as follows: cells expressing MagIC or mCherry–cpVenus, used as a negative control, were washed with -free Hanks balanced salt solution [HBSS(−); Sigma, containing, in mM: KCl, 5.33; , 0.44; NaCl, 138; , 4.0; , 0.3; glucose, 5.6; pH in the 7.2 to 7.6 range] and then the cellular membrane was permeabilized with digitonin in HBSS(−). To this, 10 mM in HBSS(−) was added, followed by the application of a saturating (20 mM) concentration of EDTA in HBSS(−). 2.10.Hyperosmotic Shock of HeLa CellsHeLa cells cultured in glass-bottom dishes for 48 h after transfection were imaged in their original medium (DMEM + 10% FBS) at 5-s interval for 10 min at room temperature. Application of the hyperosmotic shock was performed by removing the cell medium and simultaneously, a new medium with either , KCl, or d-sorbitol (Wako Chemicals, Japan) was added to obtain the equimolar final concentrations of 50 mM , 75 mM KCl, or 150 mM d-sorbitol in the imaged dish. 3.Results3.1.Indicator Design-sensitivity of the indicator was achieved following a strategy similar to that one used for the development of the low affinity indicator CatchER.12 In the present study, three of the five negatively charged residues (corresponding to 147E, 202D, and 204E in CatchER) were sequentially introduced into cp 173 Venus,18,19 becoming 220E, 31D, and 33E, respectively (in cp173Venus numeration, Table 1). Additional spontaneous mutations G4S (EGFP 177S), V89I (EGFP 18V), and Q142M (EGFP 72Q) were found in the brightest colonies expressing the -sensitive cpVenus variant. Of these, the last two were preserved in the final indicator construction. In addition, the following variants of the -sensitive cp173Venus were created: (1) with the five negative residues of CatchER, (2) with the five negative residues and reversed spontaneous mutations, and (3) with the three negative residues and reversed spontaneous mutations. However, the resulting fluorescence of all these variants in bacterial cells was dimmer (not shown). The chosen indicator variant (corresponding to the brightest colonies) displayed a variation in the fluorescence intensity as a function of the applied or concentration and constituted the intensiometric (-sensitive) part of the indicator. Unlike other single-FP based indicators (e.g., Camgaroos,20 Pericams,21 GECOs,16 or G-CaMPs22,23), in our case, the fluorescence intensity variation upon ion binding was not provoked by large intramolecular conformational changes (see details in Ref. 12). This feature led us to introduce a second FP as a constant reference, the fluorescence intensity of which did not depend on or levels, thus allowing ratiometric imaging of changes in the concentration of these ions [Fig. 1(a)]. The resulting indicator required irradiation at two separate wavelengths: one to obtain the intensiometric signal from the -sensitive Venus variant, and the other to obtain the reference signal used to perform the signals’ ratioing. 3.2.Selection of the Reference FPTo avoid undesirable resonance energy transfer between the two proteins in the indicator, FPs with excitation peaks at wavelengths longer than the emission maximum of the -sensitive Venus variant (530 nm) were selected as reference. As mentioned above, the reference fluorescence intensity should by itself be insensitive to variations in concentrations. In addition to this requirement, the reference fluorescence intensity should be large enough to reduce errors related to the stochastic character of the photon emission process, which can result in errors in the final ratio image. From the tested FPs (Table 2), the closest one to meet all the above requirements was mCherry.24 An initial approach to use photoconverted PSmOrange (excitation/emission maxima )25 resulted in almost complete absence of the Venus fluorescence due to photobleaching by the intense light used for photoconversion, without significant changes observed in the optical properties of PSmOrange (not shown). On the other hand, the nonphotoconverted PSmOrange (excitation/emission maxima ) proved the viability of the chosen approach for in vitro ratiometric imaging using a reference fluorescence signal. PSmOrange emission did not interfere with the -sensitive Venus fluorescence and could be detected separately, though this result is impractical for imaging due to low fluorescence intensity [Fig. 2(c)] and requirement of somewhat complicated filter sets in the microscopy system. In the case of other reference FPs, mKOκ,26 and mKate2,27 either Venus fluorescence was lost due to almost complete FRET [mKOκ; Fig. 2(c)] or the reference fluorescence was too dim [mKate2; Fig. 2(c)]. Moreover, the analysis of the excitation, absorption and emission spectra of PSmOrange, mKOκ, and mKate2 (in fusions with -sensitive cpVenus) revealed their sensitivity to addition of 5 mM (Fig. 2) and only mCherry spectra remained unchanged. Thus, we confirmed that mCherry fluorescence intensity is stable enough to serve as a constant reference for the indicator. Table 2Fluorescent proteins (FPs) tested as reference to obtain a ratiometric low affinity Mg2+/Ca2+-sensitive indicator. The fusion of mCherry with the -sensitive moiety described in Sec. 3.1 constituted the final indicator, termed as MagIC [Fig. 1(b)]. We should mention that during our work we found that the non-FRET ratiometric imaging approach referred to here has already been applied in several previous studies,28,29 which also used mCherry as a reference FP. In these studies, however, the performance of other reference FPs was not analyzed. 3.3.In Vitro Properties of MagICAs the reported concentration in MagIC relies on the ratio of fluorescence signals from two FPs, a preferential expression of one of them could potentially alter the results of measurements. That is why after purification, the protein samples were tested by seminative SDS PAGE to estimate their purity and the indicator integrity. For this purpose, the samples with SDS were subjected to PAGE without preliminary heating. Under these conditions, FPs retain their fluorescence and can be easily visualized on excitation using appropriate light. Our results show that more than 90% of the observed fluorescent band intensity corresponds to the correct construction in the purified protein sample [Fig. 3(a)]. First, we tested in vitro the affinity of our indicator to . The emission spectrum of MagIC displays a rising 610-nm peak with an increase in the applied concentration when the sensitive Venus variant is excited [Fig. 3(b)]. The intensity of this peak is higher than the intensity of separately excited mCherry, suggesting that it is provoked both by excitation of mCherry and the emission of the Venus variant at wavelengths longer than 580 nm. Such undesired FRET forced us to acquire the reference signal by a separate excitation, not only in vitro but also during imaging. To determine the indicator affinity for , solutions with concentrations ranging from 0 to 25 mM were used. The calculated affinity constant for this ion in the presence of 100 mM KCl at room temperature was (Hill constant ) [Fig. 3(d)] with saturation around 20 mM . The designed -chelating site does not exclude the possibility of other divalent cations binding, so the selectivity of MagIC for several of these cations was tested. As shown in Fig. 3(c), the indicator also displays affinity for in the millimolar range [, ]. This suggests that, similar to many other fluorescent dyes used for cytosolic monitoring, our indicator is also a low affinity (though of millimolar order) indicator. The selectivity of MagIC in 1 mM was analyzed against , or at a concentration of 0.1 mM each. In the presence of these concentrations, much larger than their normal levels in the eukaryotic cell cytosol, the (Venus/mCherry) ratio of MagIC remained almost unaffected. , , , and at micromolar concentrations also did not show any noticeable effect on the ratio value observed for alone [Fig. 3(e)]. The ratio value, however, was increased by the application of 1 mM spermine and spermidine, polyamines, which are known to compete with for binding sites in nucleic acids [Fig. 3(e)]. Interestingly, application of 1 mM ATP provoked a strong decrease in the (Venus/mCherry) ratio, indicating competition with MagIC for by this high affinity chelator [],30 suggesting at the same time that our indicator does not sense Mg-ATP. The of MagIC, as in the case of CatchER,12 the of MagIC is 7.5 [Fig. 3(f)], being the -sensitive Venus the main pH-sensitive component. Analysis of the kinetics of -binding by MagIC using the stopped flow method revealed values of and [at 31°C; Fig. 3(g)]. 3.4.MagIC can be Selectively Targeted to Different Intracellular CompartmentsMagIC is a genetically encoded indicator; so, in a next step, we analyzed its intracellular distribution when expressed in cells and the possibility of its targeting to specific intracellular locations. Expression of MagIC in HeLa cells without any particular targeting sequence resulted in mixed cytosolic and nuclear localization, a unique feature of our indicator [Fig. 4(a)]. MagIC-transfected cells were easily identified both by the bright yellow–green fluorescence of -sensitive Venus and by the red fluorescence of mCherry. Expression of MagIC in fusion with the targeting sequence of human ornithine transcarbamylase resulted in mitochondrial localization of the sensor [Fig. 4(a)], being it’s both components highly fluorescent at this organelle. On the other hand, MagIC targeted to ER displayed significantly reduced fluorescence of the -sensitive Venus [Fig. 4(a)]. This effect was not indicative of low level of divalent cations in the ER, as the ER-targeted negative control mCherry–cpVenus displayed even lower yellow–green fluorescence when imaged under similar conditions (not shown). A seminative SDS PAGE of the nonboiled lysate of HeLa cells expressing cytosolic MagIC displayed a single fluorescent band, indicating the expression of FPs predominantly within the full indicator construction [Fig. 4(b)]. 3.5.In Situ Sensitivity and Calibration of MagICHaving determined the in vitro properties of MagIC, we next analyzed its -sensitivity in cells. For this purpose, we initially incubated cells in solutions with increasing concentrations in the presence of ionomycin—a divalent cation ionophore. Under these conditions, MagIC displayed an increasing ratio with the increase in the extracellular concentration. However, in each case, the equilibration of the system took a relatively long time, during which the cell shape was altered, probably as a response to cytosolic elevation (not shown). In addition to these difficulties, we also found concerns in the literature regarding the use of ionophores to equilibrate concentration in cells (see in Ref. 5). To overcome this, we decided to analyze the in-cell sensitivity of MagIC using a different strategy. In the new scheme, HeLa cells expressing MagIC were treated with digitonin at the beginning of the experiment to permeabilize the cell membrane. Next, 10 mM was applied followed by the application of 20 mM EDTA. In these conditions, MagIC reported an increase in the (Venus/mCherry) ratio when was added and showed a pronounced decrease in the ratio value upon EDTA addition [Figs. 4(c) and 4(d)]. At the same time, no change in the (Venus/mCherry) ratio value was observed in the case of cells expressing mCherry–cpVenus, which was used as a negative control [Fig. 4(e)], indicating that the ratio changes observed in the case of MagIC corresponding to changes in the free concentration. 3.6.MagIC is Insensitive to Cytosolic ElevationsAs MagIC shows sensitivity to (though in the millimolar range), we next tested the possibility that its ratio value could be affected by elevations in the cytosolic levels. For this purpose, we coexpressed MagIC with the genetically encoded indicator B-GECO116 in pituitary cells, which are known to generate spontaneous transients. Simultaneous monitoring of cytosolic and dynamics in this cell line at 37°C in DMEM-F12 first showed that MagIC is insensitive to the cytosolic increase and indicated at the same time that the cytosolic dynamics was not affected by the generation of these spikes, at least during the course of imaging [Figs. 4(f) and 4(g)]. 3.7.Hyperosmotic Shock Induces MagIC Ratio Increase in HeLa CellsWith the experiments presented above, we could confirm the sensitivity of MagIC expressed in cells, however, the most demanding part of the work was to search for an appropriate stimulus that could induce visible changes in the cytosolic level. This task was further hindered by the fact that cytosolic free concentration seems to be tightly regulated even in situations when the stimulus induces a large flux through the plasma membrane.6 We initially focused on nordihydroguaretic acid (NDGA) a polyphenolic compound abundant in the creosote bush (Larrea tridentata), and which was reported to enhance the -dependent efflux mediated by the plasma membrane-located exchanger,31 and is, in addition, a potent inhibitor of the TRPM7—one of the main cellular -uptake systems.32 Application of NDGA () to cells expressing MagIC provoked an immediate and strong decrease in the (Venus/mCherry) ratio that achieved its minimum within 5 min and was thereafter maintained constant at this low level during the course of imaging [Figs. 5(a) and 5(b)]. It appeared, however, that acidification of the cytosol was largely responsible for this effect, as the negative control mCherry–cpVenus also displayed a (Venus/mCherry) ratio decrease with a very similar time course [Figs. 5(c) and (d)]. Interestingly, simultaneous application of NDGA (enhancer of exchange)31 and imipramine (inhibitor of the corresponding exchanger)31,33 slightly delayed the onset of the MagIC ratio value decrease and reduced it, then finally induced the formation of large membrane blebs within a few minutes of application [Fig. 5(e)]—an effect which was not observed when either of these chemicals was applied alone [Fig. 5(f)]. In Ref. 10, the authors noted that their genetically encoded indicator, MagFRET, did not report changes under several conditions which were previously reported to provoke increase. These conditions were (as cited in Ref. 10): (1) application of elevated in the medium,34 (2) application of as -competitor,35 and (3) perfusion with solutions containing variable concentration,36,37 each applied to different cell types. We decided to analyze which kind of response is reproduced in HeLa cells expressing MagIC in one of these conditions, namely, stimulation with high extracellular . In our experiments, treatment of HeLa cells expressing MagIC with 50 mM induced a significant MagIC ratio increase [Figs. 6(a) and 6(b)]. This result is similar to that previously observed with the -selective dye KMG-104 in HEK293T cells.34 The applied concentration (50 mM) is, however, significantly higher than the level present in physiological fluids (around 1 mM, reported elsewhere) and provoked visible shrinking of the treated cells. So, to analyze whether the MagIC ratio increase was a specific response to the high extracellular or to the associated hyperosmotic shock, we replaced by an equimolar amount of KCl or the carbohydrate d-sorbitol. Application to cells of either 75 mM KCl [Figs. 6(c) and 6(d)] or 150 mM d-sorbitol [Figs. 6(e) and 6(f)] in the medium induced a MagIC ratio increase similar to that provoked by the elevated extracellular , indicating that the presence of this last response is not indispensable for the observed effect. Part of this response seems, however, to depend on a highly probable change in the cytosolic ionic strength due to cellular water loss, since the application of these osmolytes to cells expressing the negative control mCherry–cpVenus also induced a slight (Venus/mCherry) ratio change, though in the opposite direction [Figs. 6(g)–6(m)]. To confirm that the changes observed with MagIC or the negative control mCherry–cpVenus were not associated to changes in the cytosolic pH, to which MagIC should be sensitive according to the measurements presented above [Fig. 3(f)], we performed monitoring of the cytosolic pH using a genetically encoded pH indicator, ratiometric pHluorin.17 This indicator has two absorption maxima around 410 and 470 nm and a single emission peak. The () emission ratio of pHluorin is directly proportional to the pH in its environment, so we used it for monitoring cytosolic pH under hyperosmotic stimulation (Fig. 7). We performed imaging of cells expressing pHluorin with excitation by 403.5 and 488 nm lasers lines. In this case, addition to cells of medium supplemented with 50 mM did not induce obvious changes in the cytosolic pH, indicating that the (Venus/mCherry) ratio increase reported by MagIC has a different origin. 4.DiscussionIn contrast to , whose concentration in the cell cytosol is maintained low around hundredths of nanomoles, concentration is kept relatively high in the submillimolar to low millimolar range. The concentration change in the cell cytosol during physiological processes is also different for these ions: free concentration can rapidly increase at least 10 times, while free cytosolic level is generally considered less prone to significant variations.6 Such differences in the behaviors of these ions should also influence the conditional differences in the response of their respective indicators: if for indicators a low basal fluorescence in resting conditions is desirable, such a property is difficult to achieve in -sensitive indicators of the appropriate affinity, which should be able to report small changes in the background of a relatively high concentration of this ion in the cell. Thus, contrary to , in resting conditions, a indicator with an appropriate affinity will necessarily show a nonzero signal, the intensity of which will depend on the concentration in its environment. Based on this assumption, we selected our indicator as the brightest probe from those expressed in bacterial cells. In vitro characterization of the chosen indicator, termed MagIC, proved its ability to change fluorescence properties upon increase from submillimolar concentrations up to 20 mM with a resulting relatively high dynamic range [Figs. 3(b) and 3(d)]. In FRET-based genetically encoded indicators, the signals from both FPs—the donor and acceptor—are detected simultaneously. This condition allows the monitoring of relatively fast spikes with a time resolution that depends mainly on the kinetic properties of the indicator. Free-cytosolic is also known to change under specific conditions, though this process takes several minutes,6 so we considered that the time gap between the separate acquisition of the -sensitive channel and the reference is much shorter than the occurring changes in concentrations and will not drastically affect the measurement results. The kinetic properties of MagIC [Fig. 3(g)] also correspond to those of a relatively fast indicator; its dissociation constant is around [Fig. 3(g)], approaching those of fast organic indicators with values in the 100 to range.38,39 However, the requirement of separate acquisition of both channels can potentially be a source of artifacts if the time between the acquisition of each channel is relatively long (for example, in the case of large images, slow scanning rates or fast-moving objects) and should also make the result undesirably sensitive to the mutual fluctuation of the excitation lights when separate light sources are used to obtain the signal from each of the MagIC’s components. The affinity of MagIC for is comparable to that of certain currently available -sensitive dyes [i.e., Mag-Fluo-4 with a ] with the advantage of millimolar-order affinity for [Figs. 3(c) and 3(d)] that should reduce the undesirable effects provoked by cytosolic increase, which is known to be able to affect measurements with conventional -sensitive dyes.40 Indeed, in experiments with cells capable of generating spontaneous transients, we did not observe MagIC ratio changes that could suggest an interference from cytosolic [Figs. 4(f) and 4(g)]. On the other hand, even in an object with such unusual properties, we could not find evidence of what could be a possible wave. Finally, we cannot dismiss the possibility that the low-selectivity binding site of MagIC could make our indicator sensitive to intracellular species other than cytosolic . Particularly, as we have shown in vitro, intracellular polyamines could confound the results reported by our sensor [Fig. 3(e)]. Unfortunately, in the present study, we did not have the opportunity to corroborate our results with other available techniques of monitoring, however, our results are in good agreement with the results of previous studies (e.g., Ref. 33). We, nevertheless, have shown that the cytosolic increase, reported in Ref. 33 to be induced by high-extracellular treatment, is provoked by the high osmolality of the medium rather than by itself (Fig. 6). MagIC, similarly to its prototype CatchER,12 has a relatively high sensitivity to pH [, Fig. 3(f)]. This sensitivity is probably not a big issue if monitoring is performed in the cytosol, as the pH in this compartment is rather stable41 and close to the value of MagIC. However, if measurements with our indicator are to be performed in compartments or under conditions which are supposed to affect local pH, simultaneous monitoring of this parameter could allow distinguishing pH-related effects reported by MagIC from those induced by surrounding changes. There is an available plethora of fluorescent pH indicators, either organic dyes or genetically encoded pH-sensitive proteins, which can be used to test whether a specific experimental condition induces pH changes in the monitored compartment. In conclusion, we validated the functionality of MagIC, a genetically encoded indicator based on a straightforward “modular” reporting mechanism, and we hope that it will serve as a useful tool for monitoring in different cellular compartments. AcknowledgmentsThis work was supported by the Grant-in-aid for Scientific Research on Innovative Areas, ‘Spying minority in biological phenomena (No. 3306)’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) (No. 23115003) to T. N. ReferencesA. M. P. Romani and A. Scarpa,
“Regulation of cellular magnesium,”
Front. Biosci., 5 d720
–734
(2000). http://dx.doi.org/10.2741/Romani 1093-9946 Google Scholar
Z. Grabarek,
“Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins,”
Biochim. Biophys. Acta, 1813
(5), 913
–921
(2011). http://dx.doi.org/10.1016/j.bbamcr.2011.01.017 BBACAQ 0006-3002 Google Scholar
F. Y. Li et al.,
“Second messenger role for revealed by human T-cell immunodeficiency,”
Nature, 475 471
–476
(2011). http://dx.doi.org/10.1038/nature10246 NATUAS 0028-0836 Google Scholar
A. M. P. Romani,
“Magnesium homeostasis in mammalian cells,”
Front. Biosci., 12 308
–311
(2007). http://dx.doi.org/10.2741/2066 1093-9946 Google Scholar
R. D. Grubbs,
“Intracellular magnesium and magnesium buffering,”
BioMetals, 15 251
–259
(2002). http://dx.doi.org/10.1023/A:1016026831789 BOMEEH 0966-0844 Google Scholar
A. M. P. Romani,
“Cellular magnesium homeostasis,”
Arch. Biochem. Biophys., 512 11
–23
(2011). http://dx.doi.org/10.1016/j.abb.2011.05.010 ABBIA4 0003-9861 Google Scholar
V. Trapani et al.,
“Intracellular magnesium detection: imaging a brighter future,”
Analyst, 135 1855
–1866
(2010). http://dx.doi.org/10.1039/c0an00087f ANLYAG 0365-4885 Google Scholar
B. Raju et al.,
“A fluorescent indicator for measuring cytosolic free magnesium,”
Am. J. Physiol., 256
(3), C540
–C548
(1989). AJPHAP 0002-9513 Google Scholar
H. Komatsu et al.,
“Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D imaging,”
J. Am. Chem. Soc., 126 16353
–16360
(2004). http://dx.doi.org/10.1021/ja049624l JACSAT 0002-7863 Google Scholar
L. Lindenburg et al.,
“MagFRET: The first genetically encoded fluorescent sensor,”
PLoS One, 8
(12), e82009
(2013). http://dx.doi.org/10.1371/journal.pone.0082009 1932-6203 Google Scholar
A. Sawano and A. Miyawaki,
“Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis,”
Nucl. Acids Res., 28
(16), e78
(2000). http://dx.doi.org/10.1093/nar/28.16.e78 NARHAD 0305-1048 Google Scholar
S. Tang et al.,
“Design and application of a class of sensors to monitor dynamics in high concentration cellular compartments,”
Proc. Natl Acad. Sci. U.S.A., 108
(39), 16265
–16270
(2011). http://dx.doi.org/10.1073/pnas.1103015108 PNASA6 0027-8424 Google Scholar
A. L. Horwich et al.,
“The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge,”
J. Cell Biol., 105 669
–677
(1987). http://dx.doi.org/10.1083/jcb.105.2.669 JCLBA3 0021-9525 Google Scholar
K. D. Piatkevich et al.,
“Photoswitchable red fluorescent protein with a large Stokes shift,”
Chem. Biol., 21
(10), 1402
–1414
(2014). http://dx.doi.org/10.1016/j.chembiol.2014.08.010 CBOLE2 1074-5521 Google Scholar
F. L. Graham and A. J. van der Eb,
“A new technique for the assay of infectivity of human adenovirus 5 DNA,”
Virology, 52
(2), 456
–467
(1973). http://dx.doi.org/10.1016/0042-6822(73)90341-3 VIRLAX 0042-6822 Google Scholar
Y. Zhao et al.,
“An expanded palette of genetically encoded indicators,”
Science, 333
(6051), 1888
–1891
(2011). http://dx.doi.org/10.1126/science.1208592 SCIEAS 0036-8075 Google Scholar
G. Miesenbock, D. A. De Angelis and J. E. Rothman,
“Visualizing secretion and synaptic transmission with pH-sensitive Green fluorescent protein,”
Nature, 394 192
–195
(1998). http://dx.doi.org/10.1038/28190 NATUAS 0028-0836 Google Scholar
T. Nagai et al.,
“A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,”
Nat. Biotechnol., 20
(1), 87
–90
(2002). http://dx.doi.org/10.1038/nbt0102-87 NABIF9 1087-0156 Google Scholar
T. Nagai et al.,
“Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins,”
Proc. Natl Acad. Sci. U.S.A., 101
(29), 10554
–10559
(2004). http://dx.doi.org/10.1073/pnas.0400417101 PNASA6 0027-8424 Google Scholar
G. S. Baird, D. A. Zacharias and R. Y. Tsien,
“Circular permutation and receptor insertion within green fluorescent proteins,”
Proc. Natl Acad. Sci. U.S.A., 96 11241
–11246
(1999). http://dx.doi.org/10.1073/pnas.96.20.11241 PNASA6 0027-8424 Google Scholar
T. Nagai et al.,
“Circularly permuted green fluorescent proteins engineered to sense ,”
Proc. Natl Acad. Sci. U.S.A., 98 3197
–3202
(2001). http://dx.doi.org/10.1073/pnas.051636098 PNASA6 0027-8424 Google Scholar
J. Nakai, M. Ohkura and K. Imoto,
“A high signal-to-noise probe composed of a single green fluorescent protein,”
Nat. Biotechnol., 19 137
–141
(2001). http://dx.doi.org/10.1038/84397 NABIF9 1087-0156 Google Scholar
J. Akerboom et al.,
“Optimization of a GCaMP calcium indicator for neural activity imaging,”
J. Neurosci., 32
(40), 13819
–13840
(2012). http://dx.doi.org/10.1523/JNEUROSCI.2601-12.2012 JNRSDS 0270-6474 Google Scholar
N. C. Shaner et al.,
“Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein,”
Nat. Biotechnol., 22
(12), 1567
–1572
(2004). http://dx.doi.org/10.1038/nbt1037 NABIF9 1087-0156 Google Scholar
O. Subach et al.,
“A photoswitchable orange-to-far-red fluorescent protein, PSmOrange,”
Nat. Methods, 8
(9), 771
–777
(2011). http://dx.doi.org/10.1038/nmeth.1664 1548-7091 Google Scholar
H. Tsutsui et al.,
“Improving membrane voltage measurements using FRET with new fluorescent proteins,”
Nat. Methods, 5
(8), 683
–685
(2008). http://dx.doi.org/10.1038/nmeth.1235 1548-7091 Google Scholar
D. Shcherbo et al.,
“Far-red fluorescent tags for protein imaging in living tissues,”
Biochem. J., 418
(3), 567
–574
(2009). http://dx.doi.org/10.1042/BJ20081949 BIJOAK 0264-6021 Google Scholar
Y. P. Hung et al.,
“Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor,”
Cell Metab., 14
(4), 545
–554
(2011). http://dx.doi.org/10.1016/j.cmet.2011.08.012 CMEEB5 1550-4131 Google Scholar
S. Su et al.,
“Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia,”
Nat. Methods, 10 1105
–1107
(2013). http://dx.doi.org/10.1038/nmeth.2647 1548-7091 Google Scholar
R. K. Gupta et al.,
“Measurement of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31P NMR and optical absorbance spectroscopy,”
Biochem. Biophys. Res. Commun., 117
(1), 210
–216
(1983). http://dx.doi.org/10.1016/0006-291X(83)91562-0 BBRCA9 0006-291X Google Scholar
A. Ikari et al.,
“Arachidonic acid-activated -dependent efflux in rat renal epithelial cells,”
Biochim. Biophys. Acta, 1618
(1), 1
–7
(2003). http://dx.doi.org/10.1016/j.bbamem.2003.09.011 BBACAQ 0006-3002 Google Scholar
H. C. Chen et al.,
“Blockade of TRPM7 channel activity and cell death by inhibitors of 5-lipooxygenase,”
PLoS One, 5
(6), e11161
(2010). http://dx.doi.org/10.1371/journal.pone.0011161 1932-6203 Google Scholar
F. I. Wolf et al.,
“Modulation of TRPM6 and exchange in mammary epithelial cells in response to variations of magnesium availability,”
J. Cell Physiol., 222
(2), 374
–381
(2010). http://dx.doi.org/10.1002/jcp.v222:2 JCLLAX 0021-9541 Google Scholar
H. Zhou and D. E. Clapham,
“Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development,”
Proc. Natl Acad. Sci. U.S.A., 106
(37), 15750
–15755
(2009). http://dx.doi.org/10.1073/pnas.0908332106 PNASA6 0027-8424 Google Scholar
C. P. Fonseca et al.,
“ influx and binding, and competition in bovine chromaffin cell suspensions as studied by 7Li NMR and fluorescence spectroscopy,”
Met. Based Drugs, 7
(6), 357
–364
(2000). http://dx.doi.org/10.1155/MBD.2000.357 MBADEI 0793-0291 Google Scholar
M. Schweigel et al.,
“Characterization of the -dependent transport in sheep ruminal epithelial cells,”
Am. J. Physiol. Gastrointest. Liver Physiol., 290
(1), G56
–G65
(2006). AJPHAP 0002-9513 Google Scholar
E. Murphy et al.,
“Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra,”
Proc. Natl Acad. Sci. U.S.A., 86
(8), 2981
–2984
(1989). http://dx.doi.org/10.1073/pnas.86.8.2981 PNASA6 0027-8424 Google Scholar
A. I. Escobar et al.,
“Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis,”
Pflügers Arch., 434 615
–631
(1997). http://dx.doi.org/10.1007/s004240050444 PFLABK 0031-6768 Google Scholar
M. Mank and O. Griesbeck,
“Genetically encoded calcium indicators,”
Chem. Rev., 108
(5), 1550
–1564
(2008). http://dx.doi.org/10.1021/cr078213v CHREAY 0009-2665 Google Scholar
T. W. Hurley, M. P. Ryan and R. W. Brinck,
“Changes in cytosolic interfere with measurements of cytosolic using mag-fura-2,”
Am. J. Physiol. Cell Physiol., 263
(2), C300
–307
(1992). 0363-6143 Google Scholar
Y. Bouret, M. Argentina and L. Counillon,
“Capturing intracellular pH dynamics by coupling its molecular mechanisms within a fully tractable mathematical model,”
PLoS One, 9
(1), e85449
(2014). http://dx.doi.org/10.1371/journal.pone.0085449 1932-6203 Google Scholar
BiographyVadim Pérez Koldenkova is a postdoctoral researcher at the Department of Biomolecular Science and Engineering, The Institute of Scientific and Industrial Research, Osaka University, Japan. He received his MS degree in biophysics from the Nizhny Novgorod State University, Russia, in 2003, and PhD in physiology from the University of Colima, Mexico, in 2007. His current research interests include long-distance communication in plants and application of methods of imaging for plant study. Tomoki Matsuda is an associate professor at the Department of Biomolecular Science and Engineering, The Institute of Scientific and Industrial Research, Osaka University, Japan. He received his MS and PhD from Osaka University in 1999 and 2003, respectively. His current research interests include development of genetically encoded fluorescent indicators, methods of optical analysis, and applications of fluorescent proteins. Takeharu Nagai is the principal investigator at the Department of Biomolecular Science and Engineering, The Institute of Scientific and Industrial Research, Osaka University, Japan. He graduated from Tsukuba University in 1992 and received his PhD from The University of Tokyo in 1998. So far, he has contributed to the invention of many fluorescent proteins. His research interests are expanding toward invention of tools for bioluminescence imaging, as well as microscopic methods for super-resolution functional imaging. |