©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transfected Aequorin in the Measurement of Cytosolic Ca Concentration (Ca)
A CRITICAL EVALUATION (*)

Marisa Brini (§) , Robert Marsault(§)(¶) , Carlo Bastianutto , Javier Alvarez (**) , Tullio Pozzan , Rosario Rizzuto (§§)

From the (1) Department of Biomedical Sciences and Consiglio Nazionale delle Ricerche Center for the Study of Mitochondrial Physiology, University of Padova, Padova 35121, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., [Medline] Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for measuring, in mammalian cells, the [Ca] in the cytoplasm, a compartment for which effective Caprobes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 µM) are associated with a significant buffering of the [Ca]transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca]increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca]in cotransfection experiments. In particular, we show that when cotransfected with an -adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells.


INTRODUCTION

Apoaequorin is a 21-kDa photoprotein that is linked covalently to a hydrophobic prosthetic group, coelenterazine. Upon Cabinding, aequorin undergoes an irreversible reaction, with production of light in the visible range. The fractional rate of aequorin consumption is proportional, in the physiological pCa range, to [Ca]. Because of this relationship, the purified photoprotein, microinjected in living cells, has been employed for more than 3 decades as a Caindicator (4, 5, 6, 7) .

The need for microinjection or other traumatic means of cell loading has limited the use of this indicator to large and robust cells. Thus, in the last decade, aequorin has been substituted largely by fluorescent Caindicators, such as quin2, fura-2, indo1, etc., which can be loaded easily in virtually all cell types via their intracellularly trappable esters (8, 9) . Recently, however, the cloning of the aequorin cDNA (10, 11) has expanded the possible uses of this Ca-sensitive photoprotein. First, the possibility of expressing the transfected gene recombinantly has eliminated the need for traumatic loading procedures, by allowing the endogenous production of the photoprotein in cell systems as diverse as bacteria (12) , yeasts (13) , slime molds (14) , plants (15) , and mammalian cells (16, 17) . Second, the aequorin cDNA can be engineered to include defined targeting signals, thus determining the specific localization of the Caprobe inside the cell (1, 2, 3, 18, 19, 20, 21, 22) .

Recombinant apoaequorin can be reconstituted into active photoprotein by simply adding the coenzyme to the incubation medium; aequorin light emission then allows the monitoring of the [Ca] in the compartment to which the photoprotein is confined. For subcellular organelles, the possibility of specific targeting confers to recombinant aequorin a clear advantage over the alternative methods presently available ( e.g. fluorescent dyes). Similarly, in various cell types (12, 13, 14, 15) , the difficulty in loading fluorescent indicators has made recombinant aequorin the method of choice for monitoring [Ca].() On the contrary, in mammalian cells the possible advantages of recombinantly expressed aequorin, first reported by Button and Brownstein (16) and Sheu et al. (17) , are not obvious. Only a direct comparison between the available Caprobes may highlight the conditions in which recombinant aequorin can complement, or substitute, the other techniques. To this end, we report here the development of a new cDNA construct that encodes an epitope-tagged aequorin molecule (which can be easily immunolocalized with specific antibodies); the encoded polypeptide is located in the cytosol and allows the monitoring of [Ca]in transiently and stably transfected cells. We also provide a comprehensive picture of the methodology (and of the procedure for calibrating the luminescence data into [Ca]values) and a direct comparison between the data obtained, in the same cells, with recombinant aequorin and fura-2.


MATERIALS AND METHODS

Construction of cytAEQ

To include a strong immunological epitope, the coding region of the aequorin cDNA (11) was amplified by PCR using specifically modified oligonucleotide primers. In particular, the upstream primer included a start codon, a HindIII site, and the nucleotides encoding the nine amino acids of the HA1 hemagglutinin epitope (23) and amino acids 2-9 of aequorin. The reverse primer corresponded to a sequence located in the 3`-noncoding region (spanning the authentic EcoRI site of the aequorin cDNA). The PCR product was inserted in the SmaI site of pBS+ (Stratagene) and controlled by DNA sequencing; the whole coding sequence was then excised by double digestion with PstI (a flanking vector site) and EcoRI and subcloned in the eukaryotic expression vectors pMT2 (24) and pcDNAI (InVitrogen). The recombinant vectors allow the expression of a simply modified aequorin molecule that includes only the epitope in front of the photoprotein (cytAEQ). A silent mutation was introduced in the native HindIII site of the aequorin cDNA, so by digesting the PCR product with HindIII and EcoRI, a fragment can be released which encodes the HA1 epitope and aequorin; this cDNA was also employed for the construction of specifically targeted epitope-tagged aequorins.()Fig. 1 shows the sequence of the upstream sense primer used for the PCR and a schematic map of the wild type (pAQ440) (11) and PCR-modified (cytAEQ) aequorin cDNAs.


Figure 1: Construction strategy of cytAEQ. On the right, the PCR strategy employed for constructing cytAEQ is summarized. In the schematic maps of the parental aequorin cDNA clone and of the cytAEQ PCR product, the noncoding portions of the cDNAs are represented as lines; dark and light boxes indicate the portions of the cDNAs encoding the epitope and the photoprotein, respectively. The positions of the relevant restriction sites and of the start codon ATG are also indicated. Arrows indicate the positions of the PCR primers. The downstream antisense primer corresponds to nucleotides 641-662 of the aequorin cDNA (11); the complete sequence of the upstream sense primer (which includes nucleotides 24-51 of the aequorin cDNA) is shown in detail in the left part of the figure since it specifies the modifications introduced in the aequorin cDNA. pAQ440, wild type aequorin cDNA (11); cytAEQ, PCR-modified aequorin cDNA.



Cell Culture and Transfection

HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 75-cmFalcon flasks. For the transient expression of cytAEQ, the cells were plated onto 13-mm diameter round coverslips, and transfection with 4 µg/well of plasmid DNA was carried out as described (25) . Aequorin measurements were carried out 36 h after transfection, as described (19, 21) .

HeLa clones stably expressing cytAEQ were generated by cotransfecting the cytAEQ expression vector with the pSV2neo plasmid (26) and selecting G418-resistant clones, as described (19) .

Primary cultures of rat embryonic cortex neurons were obtained as reported previously (27) ; the cells were transfected with cytAEQ on the 4th day of culture, and aequorin measurements were performed as described above.

Aequorin Reconstitution and Measurement

Transfected aequorin was reconstituted by incubating the cells for 2 h with 5 µM coelenterazine in Dulbecco's modified Eagle's medium + 1% fetal calf serum, at 37 °C in 5% COatmosphere and aequorin light emission were measured in a purpose-built luminometer (19, 21, 28) .

In Vitro Calibration of cytAEQ

The calibration curve of cytAEQ was determined in vitro by exposing cell lysates of cytAEQ-expressing HeLa cells to solutions with known [Ca]. For this purpose, HeLa cells were transiently transfected with cytAEQ on a 10-cm Petri dish, as described (25) ; 36 h after transfection, the cells were washed twice with PBS, scraped with a rubber policeman into 200 µl of a solution containing 150 mM Tris, 0.8 mM phenylmethylsulfonyl fluoride, 0.1 mM EGTA, pH 7.0, and lysed through three cycles of freeze-thawing. After centrifuging for 5 min at 4 °C at 12,000 rpm (Eppendorf microcentrifuge), the cell pellet was discarded. The supernatant (cell lysate) was utilized for the experiments, after reconstituting apoaequorin with 5 µM coelenterazine for 2-3 h at 4 °C, in the presence of 140 mM -mercaptoethanol, as described (25) . The various Cabuffers were prepared by supplementing a saline solution (130 mM KCl, 10 mM NaCl, 1 mM MgSO, 0.5 mM KHPO, 20 mM Hepes, pH 7.0, at 22 °C) with 5 mM EGTA (or HEDTA for the [Ca] higher than 2 µM) and different amounts of total CaCl. The free Caconcentration of each buffer was calculated by a computer program based on the Caaffinity constants of EGTA and HEDTA, as described by Fabiato (29) . For [Ca] lower than 2 µM, the [Ca] of the buffers was also measured with the fluorescent Caindicator fluo-3. In the experiment, 10 µl of the cell lysate was transferred to the sample chamber of the luminometer, light emission recording was started, and 90 µl of the Cabuffers was injected in the chamber. After 1 min, 2.5 µl of a 100 mM CaClsolution was injected, and recording was continued until all aequorin was consumed, i.e. until light emission returned to basal values. The count number of the sample ( L) was that measured immediately after the addition of the Cabuffer, and the total number of counts ( L) was the integral of counts from that moment to the end of the experiment. All experiments were performed at 22 °C. Based on the experimental data, the cytAEQ calibration curve was obtained as explained in the text.

Fura-2 Measurements

[Ca]measurements with fura-2 were performed on monolayers of HeLa cells as described previously (18, 19) .

Immunolocalization of cytAEQ

36 h after transfection, HeLa cells were fixed with 3.7% formaldehyde in PBS for 20 min, washed two or three times with PBS, and then incubated for 10 min in PBS supplemented with 50 mM NHCl. Permeabilization of cell membranes was obtained with a 5-min incubation with 0.1% Triton X-100 in PBS, followed by a 30-min wash with 0.2% gelatin (type IV, from calf skin) in PBS. The cells were then incubated for 1 h at 37 °C in a wet chamber with a 1:400 dilution in PBS of the anti-HA1 monoclonal antibody 12CA5 (kind gift of J. Pouyssegur, Nice, France). The binding of the antibody was revealed with a rhodamin-labeled anti-mouse IgG antibody. After each antibody incubation, the cells were washed three to five times with PBS. Fluorescence was then analyzed with a Zeiss Axioplan microscope and photographed with a Kodak Technical Pan film.


RESULTS

Intracellular Localization of the Recombinant Protein

The expression of the recombinant protein and its intracellular localization were verified by immunocytochemistry using a monoclonal antibody to the HA1 epitope. HeLa cells were transfected with the cytAEQ expression plasmid and immunostained with the antibody 36 h after transfection. Fig. 2shows the bright-field image of the cell monolayer (Fig. 2 A) and the corresponding fluorescence image (Fig. 2 B). A subset of transiently transfected cells was clearly immunopositive. The percentage of transfected cells was approximately 10-20%, similar to what was observed with other aequorin plasmids ( e.g. mitochondrially targeted or nuclear targeted aequorin expression vectors; not shown). The level of expression was quite variable from cell to cell, as evident in Fig. 2, which includes three positive cells, two strongly and one very weakly labeled. The immunolabeling is diffused through the whole cell body, up to the fine processes. In cells expressing a lower level of recombinant aequorin (in the center and on the right), it is apparent that the nuclear region is more weakly stained, suggesting exclusion from the nucleoplasm; in the rest of the cytosol a granularity can be observed, particularly evident in the regions rich in organelles. Confirming that the expressed protein is confined to the cytosol, a complete release of aequorin, with a time course indistinguishable from that of a cytosolic marker, lactic dehydrogenase, was observed upon digitonin permeabilization of the plasma membrane (data not shown and see Ref. 2). To estimate the concentration of the recombinant protein expressed by the cells, and thus its Ca-buffering capacity (see below), we measured the aequorin light output after lysing a coverslip of cells with a Ca-rich hypotonic solution and deduced the aequorin content by comparison with the light emission of known amounts of purified photoprotein. The aequorin content was estimated to be about 0.26 ng of aequorin/mg of total protein. Considering that the expressed photoprotein is restricted to the transfected cells, the real content of this subset of cells can be considered to be about 1-2 ng/mg of protein, corresponding to a cytosolic concentration of 10 M.


Figure 2: Immunolocalization of cytAEQ. Panel A shows the phase-contrast image of the cell monolayer transiently transfected with the cytAEQ cDNA, and panel B shows the corresponding fluorescence staining with the anti-HA1 antibody. Bar, 40 µm.



Calibration of the Aequorin Signal

To transform luminescence values into [Ca] values we have used the method described by Allen and Blinks (6) , which relies on the relationship between [Ca] and the ratio between the light intensity recorded in physiological conditions ( L, counts/s) and that which would have been recorded if all of the aequorin in the cell had been suddenly exposed to a saturating [Ca] ( L). Given that the rate constant of aequorin consumption at saturating [Ca] is 1.0 s(6) , a good estimate of Lcan be obtained from the total aequorin light output recorded from the cells after discharging all of the aequorin. This usually requires the addition of excess Caand detergents, or other permeabilization procedures (see below), at the end of the experiment. As aequorin is being consumed continuously, the value of Lis not constant and decreases steadily during the experiment. The value of Lto be used for [Ca] calculations at every point along the experiment should be calculated as the total light output of the whole experiment minus the light output recorded before that point.

The relationship between the ratio ( L/ L) and [Ca] has been modeled mathematically (30) . The model preferred by the authors (model B) postulates that each of the Cabinding sites has two possible states, T and R, and light is emitted when all of the sites are in the R state. Cais assumed to bind only to the R state and therefore shifts the equilibrium in favor of the R states. This model contains three parameters: K, the Caassociation constant; K= [T]/[R]; and n, the number of Cabinding sites. Experimental data obtained with native aequorin solutions in 150 mM KCl, pH 7.0, and 22 °C were perfectly fitted using values of K= 7 10 M, K= 118, n = 3 (30) . Given that these values were obtained with native aequorin (a mixture of several protein isoforms bound to natural coelenterazine), while cytAEQ represents a single isoform, modified at the amino terminus and reconstituted with a chemically synthesized coelenterazine, we thought it necessary to determine the K, K, and n values from the recombinantly expressed recombinant photoprotein. Fig. 3shows a calibration of the cytAEQ luminescence/[Ca] relationship. Experimental data were obtained by mixing a solution containing cytAEQ with solutions containing different [Ca], prepared to give defined pCa values in the final solution. Then, both the count number obtained immediately after the mixing and the total number of counts in the sample were measured to obtain L/ Lratios. These data pairs, final pCa and L/ Lratios, plotted in Fig. 3 , were then used to fit a theoretical curve based on the model mentioned above, obtaining the best values for the parameters K, K, and n. Fitting was made by using a computer routine designed to search for the values of these parameters which minimize the sum [pCa(exp) pCa(theor)]extended to all of the experimental data, where pCa(theor) are the pCa values calculated from the model (see Equation 1) when using the ratios L/ Lcorresponding to each experimental pCa(exp) value. The values we obtained in this way ( K= 7.23 10 M, K= 120, n = 2.99) for the fitting of the [Ca]/( L/ L) relationship of recombinant aequorin are very similar to those of Allen and co-workers (30) . Therefore, and no matter if the model is correct at the molecular level or not, it can confidently be used to transform luminescence values obtained with recombinant aequorin into [Ca] values. A straightforward reordering of the equation for model B reported by Allen et al. (30) provides the algorithm we use to calculate the [Ca] values at each point

 

On-line formulae not verified for accuracy

where ratio = ( L/ L), and K, Kand n are the parameters of the model. These parameters should be obtained as the best fit of the model to a calibration curve performed in conditions as close as possible to the experimental ones, particularly in terms of [Mg] and ionic strength (31, 32) . Regarding the temperature, an increase from 22 to 37 °C produces essentially a shift of about 0.15 log[Ca] units to the left in the log L/ L versus log[Ca] plot, apart from some minor effects at very high and at very low [Ca] values (32) . If this correction is taken into account, a calibration made at 22 °C can be used for calculating [Ca] values of experiments performed at 37 °C. In terms of the parameters of the model, the correction is achieved by multiplying Kby 1.4, leaving untouched the other parameters. Aequorin-based Measurement of [Ca]-For the monitoring of [Ca], HeLa cells were transfected with cytAEQ; 36 h after transfection, aequorin was reconstituted by adding the prosthetic group to the incubation medium as described under ``Materials and Methods.'' This simple procedure allowed the reconstitution of aequorin in a large number of cell types, ranging from the slime mold Dictyostelium discoideum to various mammalian cell lines or primary cultures (data not shown), implying that the diffusion of the prosthetic group across cell membranes does not represent, in the use of cytAEQ, a major problem. After reconstitution, the coverslip with the cells was transferred to the measuring apparatus, and aequorin light output was monitored. Fig. 4shows the luminescence data, at rest and upon stimulation with two inositol 1,4,5-trisphosphate-generating agonists, histamine and ATP, and their calibration into [Ca]values. At rest, the light output was close to the background levels (4-8 counts/s versus 2-3). Upon stimulation, a sharp increase could be noticed in aequorin light emission, followed by a slowly declining sustained plateau (Fig. 4 A). At the end of the experiment, unconsumed aequorin was discharged by osmotically lysing the cells with a 10 mM CaClsolution (19) . By this means, as soon as the photoprotein is released in the chamber containing high [Ca] and/or [Ca]is increased, all photons are emitted and collected by the photomultiplier. Lcorresponds to the total light output collected during the experiment, subtracted of the background luminescence ( i.e. that measured with a coverslip of nontransfected cells loaded with coelenterazine).


Figure 3: Calibration of cytAEQ luminescence. 10 µl of the cytAEQ cell lysate was mixed with 90 µl of the saline solution containing buffered [Ca] (see ``Materials and Methods'' for details). L, light emission immediately after mixing; L, integral of aequorin counts from the mixing to the end of the experiment ( i.e. after aequorin consumption with excess Ca). The continuous curve corresponds to the best fit of the experimental data to the model B of Ref. 30, as detailed under ``Results.''




Figure 4: Light emission and calculated [Ca] values in coverslips of HeLa cells transiently expressing cytosolic aequorin. The cells were trypsinized, plated on round glass coverslips, transfected with cytAEQ, and left in culture for 2 days, as described under ``Materials and Methods.'' Reconstitution with coelenterazine, detection, and calibration of the luminescence signal were carried out as described under ``Materials and Methods.'' Medium (modified Krebs-Ringer buffer): 125 mM NaCl, 5 mM KCl, 1 mM NaPO, 1 mM MgSO, 1 mM CaCl, 5.5 mM glucose, 20 mM Hepes, pH 7.4, 37 °C. Where indicated, the cells were stimulated with 100 µM histamine ( hist.) or 100 µM ATP. Panel A shows the light emission of cytAEQ (in counts/s ( cps)); panel B shows the calculated [Ca] values, obtained with the algorithm described under ``Results.'' These and the following traces are typical of more than five similar experiments, which gave the same results.



The absolute values of the luminescence peaks were dramatically different with the two agonists: 12,000 and 2,000 counts/s with histamine and ATP, respectively. However, when the crude luminescence data were converted into [Ca]values (Fig. 4 B), the difference appeared much smaller (1.7 and 0.9 µM at the peak with histamine and ATP, respectively). It is thus clearly apparent that, as expected from the nonlinear response curve of aequorin and the progressive consumption of the indicator throughout the experiment, the crude luminescence data largely amplify the differences in [Ca]. The presentation of the aequorin light signal can thus be misleading: the aequorin signal must be calibrated not only for providing accurate estimates of the absolute [Ca]values but also for obtaining a correct qualitative picture of the [Ca]changes.

Fura-2 and cytAEQ: A Direct Comparison

To compare directly the [Ca]data obtained with fura-2 and cytAEQ, HeLa cells were transfected with cytAEQ and loaded with fura-2, and [Ca]was measured with the two indicators in parallel batches of cells. Unlike the situation of Fig. 4, in the experiment presented in Fig. 5the cytAEQ signal was recorded from cells also loaded with fura-2. It is apparent that the [Ca]estimates are quite similar with the two probes cytAEQ and fura-2, indicating that the two probes, although different in nature and thus requiring distinct calibration procedures, provide a coherent display of the [Ca]changes occurring in the cytosol of eukaryotic cells. The peak rise, as monitored with cytAEQ, appeared somewhat wider compared with that revealed by fura-2. Probably this minor difference reflects the faster and more homogeneous mixing of the solution in the latter type of experiment (see Fig. 5legend). Compared with Fig. 4, the peak rise in [Ca], as measured with aequorin, was quite reduced in amplitude (1.3 versus 1.7 µM). In five similar experiments, the peak [Ca]values measured by cytAEQ were 1.7 ± 0.1 and 1.25 ± 0.15 in cells without and with fura-2, respectively. The simplest explanation of this difference is the extra Cabuffering provided by the fluorescent Caindicator.


Figure 5: Direct comparison of [Ca] data obtained with fura-2 and cytAEQ. HeLa cells were both transiently transfected with cytAEQ and loaded with fura-2. [Ca] was measured with the two indicators in different coverslips of cells, as described under ``Materials and Methods.'' The upper and lower traces show the [Ca] changes measured with fura-2 and cytAEQ, respectively. Where indicated, the cells were challenged with 100 µM histamine. In the case of fura-2 experiments ( upper panel), 20 µl of a concentrated histamine solution was added to the cuvette under continuous stirring. Mixing time under these conditions was 1 s. In the aequorin measuring apparatus (19, 21, 28) ( lower panel), histamine was added to the medium and reached the cells through the perfusion system. In this latter case the mixing time in the measuring chamber was slower than in the cuvette (2-3 s). These differences in the application of the stimulus probably account for the small differences in the sharpness of the [Ca] peaks with the two probes. Other conditions are as in Fig. 4.



The buffering effect of fura-2 loading was investigated directly in the experiment presented in Fig. 6, in which the cells were loaded with a standard dose of fura-2/AM (3 µM), and fura-2 concentration and [Ca](monitored with cytAEQ) were simultaneously measured in parallel batches of cells. The dashed and solid lines refer to unloaded cells and cells containing 50 µM fura-2, respectively. Even with this intracellular fura-2 concentration, the peak [Ca]increase was reduced significantly when compared with control cells (1.7 versus 1.3 µM), although the following sustained plateau was not affected (see ``Discussion''). In Double Transfection Experiments, cytAEQ Selectively Measures [Ca] in the Subset of Transfected Cells-When a protein known to be involved in calcium signaling ( e.g. a channel, a receptor, or a calcium-binding protein) is transiently expressed in a population of cells, a major goal is the monitoring of Cahomeostasis in the subset of cells that express the foreign protein. Although other solutions are certainly possible (see ``Discussion''), the use of cytAEQ takes advantage of the fact that when two cDNAs are cotransfected, the encoded proteins are expressed in the same subset of cells (33) .


Figure 6: Measurement, with cytAEQ, of [Ca] changes in fura-2-loaded and control HeLa cells. The cells, transiently transfected with cytAEQ, were loaded with a standard dose of fura-2/AM (3 µM). Fura-2 was measured in the supernatant after permeabilization of the cells with 100 µM digitonin, and the intracellular concentration calculated by comparison with a standard of fura-2-free acid. [Ca] was monitored with cytAEQ, as described in Fig. 4. The dashed and solid lines refer to unloaded cells and cells containing 50 µM fura-2, respectively. Where indicated, the cells were stimulated with 100 µM histamine; other conditions are as in Fig. 4.



In the experiment presented in Fig. 7, HeLa cells were transfected with both the cytAEQ cDNA and that encoding the -adrenergic receptor, which is coupled via a Gprotein to inositol 1,4,5-trisphosphate generation. Having no endogenous -adrenergic receptor, HeLa cells transfected with only cytAEQ responded with a [Ca]rise, both with fura-2 and cytAEQ, upon histamine, but not norepinephrine, challenge (Fig. 7, a and b). When the cells were also transfected with the -adrenergic receptor, fura-2 and cytAEQ revealed, upon norepinephrine stimulation, two dramatically different pictures. Using cytAEQ (Fig. 7 d), a large [Ca]rise (2 µM at the peak) could be observed upon norepinephrine challenging, which was in the range of those observed in fura-2-loaded responsive cells, analyzed at the single cell level with an imaging apparatus (not shown). The following stimulation with histamine evoked a much smaller [Ca]rise, as expected since the intracellular Castores had been largely emptied in the norepinephrine-responsive cells. Conversely, when the whole cell monolayer was monitored with fura-2 (Fig. 7 c), the signal originated mainly from nontransfected cells, and thus the effect of norepinephrine was almost negligible, whereas the following histamine-induced [Ca]rise was only slightly reduced, when compared with that of control cells (Fig. 7 a).


Figure 7: Measurement of [Ca], with cytAEQ and fura-2, in cells transfected with the -adrenergic receptor cDNA. Traces a and b, control cells, transfected with cytAEQ only. Traces c and d, cells transfected with both cytAEQ and -adrenergic receptor cDNAs. a and c, fura-2; b and d, cytAEQ. Where indicated, the cells were challenged with 100 µM histamine ( hist.) or 100 µM norepinephrine ( norep.). Other conditions are as described in Figs. 4 and 5.



CytAEQ Can Be Expressed in a Variety of Transiently or Stably Expressed Cell Types

All of the data presented so far refer to the transient expression of cytAEQ in the cell line HeLa. This is obviously not a peculiarity of this cell line, as we successfully transfected cytAEQ in a variety of other cell lines of different embryological origin (data not shown).

Figs. 8 and 9 shows two other extensions of the approach. Fig. 8shows the [Ca]monitoring with cytAEQ in primary cultures of neurons, transiently expressing the recombinant photoprotein. The high level of expression allowed also in this case an accurate conversion of luminescence data into [Ca]values. Interestingly, in these cells the depolarization-dependent opening of the plasma membrane Cachannels caused a very large increase in mean [Ca](peaking at approximately 3.5 µM), which was well detected by the photoprotein, since the saturation of the aequorin signal occurs at > 10 µM.


Figure 8: [Ca] monitoring with cytAEQ in primary culture of neurons. The neuronal culture was obtained and transfected with cytAEQ, as described under ``Materials and Methods.'' Where indicated, the cell monolayer was perfused with a KCl-rich medium (120 mM KCl, 5 mM NaCl, 1 mM NaPO, 1 mM MgSO, 1 mM CaCl, 5.5 mM glucose, 20 mM Hepes, pH 7.4, 37 °C). Other conditions are as in Fig. 4.



Fig. 9 shows the [Ca]monitoring in a HeLa clone stably expressing cytAEQ. Numerous cytAEQ-expressing clones were generated by cotransfecting into HeLa cells the cytAEQ expression plasmid together with the selectable plasmid pSV2neo and selecting the positive clones with G418. The clone utilized in this experiment (CH5) retains the agonist sensitivity of the parental line and indeed undergoes a large [Ca]increase (peaking at approximately 1.1 µM) upon stimulation with histamine. Stably expressing clones may be useful in cell physiology studies because they provide a simple, consistent cell system, loaded with the indicator with no need to undergo even the nontraumatic procedure of cell transfection.


Figure 9: [Ca] monitoring in a HeLa clone stably expressing cytAEQ. Where indicated the cells were stimulated with 100 µM histamine. Other conditions are as in Fig. 4. CH5 HeLa clone, cytosolic HeLa 5 clone, obtained as described under ``Materials and Methods.''




DISCUSSION

A major limitation to the use of the Ca-sensitive photoprotein aequorin has been the need for introducing the polypeptide in living cells, which usually required traumatic procedures, such as microinjection or scrape loading. We show here that the possibility of expressing the protein with molecular biology techniques largely circumvents this problem: by this means, we have nontraumatically loaded with aequorin all of the cell types we tested, which include both cell lines (HeLa, L929, ins-1) and primary cultures (neurons, myocytes). In the former case, clones constitutively expressing the Caindicator can also be selected. On average, although a clear cell heterogeneity is observed in transient expressions, the photoprotein is expressed at sufficiently high levels to allow an accurate calibration of the luminescence data into [Ca]values. As to the calibration procedure, our data confirm previous observations that recombinant aequorin retains the Casensitivity of native aequorin (34, 35) and indicate that additions to the amino terminus do not significantly modify the Caaffinity of the photoprotein. We have thus developed a computer algorithm for converting off-line the aequorin luminescence data into [Ca] values, which is based on the Caresponse curve of cytAEQ at physiological conditions of pH, temperature, ionic strength, and [Mg].

A first clear observation is that in fura-2-loaded cells, the [Ca]estimates of cytAEQ, both at rest and upon stimulation, closely match those of fura-2, although, as expected from the steep Caresponse curve of aequorin, the calibrated signal at low [Ca] appears somewhat noisy. The two probes appear to give a coherent picture of the [Ca]changes, and thus a choice can be made uniquely based on their specific characteristics, which may render them appropriate to different applications. Intracellularly trappable fluorescent dyes (8, 9) have become, in the last decade, the most commonly used Caprobe in intact cells. Because of the high Caselectivity, the strong signal (which allows population and single cell measurement), and the ease of use, in most cases they should be regarded as the first choice. Moreover, aequorin calibration requires the lysis of the cells at the end of the experiment (thereby excluding the possibility of other applications at the end of the [Ca]measurement). Coelenterazine analogues are becoming available, however, which allow a ratiometric calibration also of aequorin (36) . Finally, some of the possible applications still need to be worked out for the recombinant photoprotein. In particular, the low light emission of aequorin does not allow, for the moment, the use of cytAEQ for single cell analyses of Cahomeostasis with traditional apparatuses. However, when imaging systems designed for low light collection are employed (37) , transfected aequorin can be employed also for this type of experiments()(36) .

A few situations can, however, be envisaged in which the use of cytAEQ can provide significant advantages. A first point regards the intracellular localization. It is common knowledge that the fluorescent dyes, when loaded via the ester form, are mostly located in the cytosol but can also be sequestered by intracellular organelles (38) . This phenomenon, which is highly variable in the different cell types, can largely complicate the interpretation of the results, and often confocal microscopy and/or mathematical deconvolution algorithms need to be employed to solve the issue. The transfected protein does not face this problem, since in the absence of specific targeting signals, aequorin is translated in the free cytosolic ribosomes and is localized exclusively in the cytosol of the cells. Similarly, the transfected photoprotein allows us to solve another problem frequently encountered with the dyes, i.e. the difficulty of loading and/or the leaking out of the cells. On the former, we and others have observed that D. discoideum, which is totally resistant to loading by acetoxymethylesters of fluorescent Caindicators, can be easily transfected with cytAEQ, thereby allowing the monitoring of [Ca]()()(14) . On the latter, we observe no loss of the transfected protein in experiments for time periods as long as several hours.

Another major drawback of the intracellularly trappable fluorescent dyes has been the fact that to provide a strong signal (well above the endogenous fluorescence of the cells), they must be loaded at relatively high concentrations. Concentrations as high as 500 µM-2 mM were necessary for the first dye that was developed, quin2, whereas fura-2 and the other dyes of newer generation usually reach an intracellular concentration in the range of 20-200 µM. Conversely, the concentration of microinjected aequorin is approximately 1 µM, whereas that of the recombinantly expressed protein can be estimated to be about 1 order of magnitude lower. In both cases the cells are loaded with a molecule interfering with cellular Cahomeostasis (an EGTA derivative in the former case, a Ca-binding protein in the latter). However, because of the enormous difference in concentration, their perturbing effect on [Ca]is expected to vary significantly.

The experiments shown in this article indicate that this is indeed the case. Large, agonist-induced [Ca]rises were observed with cytAEQ, with peak values of approximately 1.5-2 µM. No significant difference was observed when the level of expression of the protein was decreased 10-fold (although in this case the noise of the trace was obviously significantly increased), suggesting that the Cabuffering effect of the photoprotein is negligible. In contrast, when the cells were loaded with fura-2, also at moderate concentrations (50 µM), a clear decrease was observed in the amplitude of the agonist-induced [Ca]peak, indicating that even at this concentration the fluorescent dye causes a significant reduction of the agonist-induced [Ca]peak. The two components of the [Ca]rise (release from intracellular stores and prolonged influx from the extracellular medium) are not equally affected. In the former case, a limited amount of Cais released, and in fact the increase of the Cabuffering capacity of the cell invariably reduces the peak rise of [Ca](which largely depends on the release of Cafrom intracellular stores). Conversely, in the case of Cainflux form the external medium, a practically unlimited reservoir of Cais available, and the steady-state [Ca]level depends solely on the relative rates of Cainflux and Caextrusion. Indeed, fura-2 loading causes no change in the plateau phase of the [Ca]rise, dependent on the prolonged opening of plasma membrane channels.

It can thus be envisaged that to introduce the minimal artifactual quantitative or qualitative changes in the Cadynamics, cytAEQ may represent the method of choice, at least in a population of cells. The amplitude of the [Ca]increase has often been reported to be critical for the induction of the cell response (39) , and the possibility of not interfering with this parameter appears of utmost interest in the study of the physiological effect of the Casignal. To this end, aequorin appears particularly well suited also because of its wide dynamic range, which allows the detection, with the same probe, of [Ca]increases ranging from approximately 0.3 µM to >10 µM (see, for example, the amplitude of the [Ca]increase in the experiment with neuronal cells presented in Fig. 8). The lack of Cabuffering effect and the wide dynamic range are not the only methodological advantages that the transfected protein may provide. When the cells are loaded with the ester form of the fluorescent dyes, hydrolysis products are released in the cells, which in some cell types may significantly alter their physiological response (40) . When these effects need to be avoided, the transfected photoprotein may represent an appealing alternative.

Finally, cytAEQ appears ideally suited for monitoring the Cachanges that occur in cells transfected with other genes. The possibility of selectively altering the molecular repertoire of a cell has become a powerful tool in cell biology. In particular, channels, receptors, and other proteins involved in Casignaling have been expressed in a variety of cell types, with the goal of studying their function and/or their coupling mechanisms. [Ca]is a key parameter to be monitored in this type of studies, and fluorescent dyes have traditionally been employed. However, when clones are generated which express a foreign protein, functional data rely on the comparison with the parental line and/or control clones, and the high variability between cell clones complicates the interpretation of the results. On the other hand, when the cells are transiently transfected, there is obviously no way to load the dye selectively in the transfected cells (which usually represent about 10-20% of total); the signal originates from the whole cell population, and the effect of the gene manipulation can be easily overlooked. Single cell imaging of [Ca]must therefore be performed, and a large statistical analysis is necessary for minimizing variabilities in cell responses. In contrast, cytAEQ can be cotransfected with the gene of interest, taking advantage of the fact that in the commonly employed transfection protocols, the DNA is taken up by the same subset of cells. By this means, a simple way is available for averaging [Ca]analysis over the whole pool of transfected cells.

In conclusion, the cloning of the aequorin cDNA has largely extended the use of this Caprobe to all cells amenable to transfection. In all the cases investigated by us, a sufficient amount of photoprotein can be expressed to allow an accurate [Ca]monitoring. The possibility of generating transgenic animals expressing cytAEQ (or other aequorin chimeras) should allow us to obtain primary cultures of mammalian tissues constitutively loaded with a Caindicator and, possibly, also to use this probe for studies in situ.


FOOTNOTES

*
This work was supported in part by grants from the Italian Research Council (CNR), Biotechnology and Oncology; Telethon; the Italian Association for Cancer Research (AIRC); the AIDS project of the Italian Health Ministry; the Italian Ministry of University and Scientific Research; and the British Research Council (to T. P. and R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors equally contributed to this work.

Recipient of a European Union Human Capital and Mobility fellowship.

**
Supported by a fellowship of the Spanish Direccion General de Investigation Cientifica y Tecnica.

§§
To whom correspondence should be addressed: Dept. Biomedical Sciences, University of Padova, Via Trieste 75, 35121 Padova, Italy. Tel.: 39-49-828-6569; Fax: 39-49-828-6576; E-mail: rizzuto@cribi1.bio.unipd.it.

The abbreviations used are: [Ca], cytosolic calcium concentration; PCR, polymerase chain reaction; cytAEQ, cytosolic aequorin; PBS, phosphate-buffered saline; HEDTA, N-hydroxyethylethylenediaminetriacetic acid.

R. Marsault, M. Brini, T. Pozzan, and R. Rizzuto, in preparation.

L. Jaffe and G. Rutter, personal communication.

L. Jaffe, personal communication.

M. Brini, R. Rizzuto, and T. Pozzan, unpublished data.


ACKNOWLEDGEMENTS

We thank G. Ronconi and M. Santato for technical assistance; Y. Sakaki and S. Cotecchia for the aequorin and -adrenergic receptor cDNAs, respectively; J. Pouyssegur and Y. Kishi for the gift of samples of the anti-HA1 monoclonal antibody 12CA5 and of coelenterazine, respectively; P. Cobbold for help in constructing the aequorin detection system; and G. Carmignoto, C. Fasolato, B. Innocenti, M. Murgia, and M. Montero for helpful discussion.


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