From the
Targeted recombinant aequorins represent to date the most
specific means of monitoring [Ca
Apoaequorin is a 21-kDa photoprotein that is linked covalently
to a hydrophobic prosthetic group, coelenterazine. Upon Ca
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
Ca
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
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.
The
relationship between the ratio ( L/ L
On-line formulae not verified for accuracy
where ratio =
(
L/
L
Figs. 8 and 9 shows
two other extensions of the approach. Fig. 8shows the
[Ca
A major limitation to the use of the
Ca
A first clear observation is that
in fura-2-loaded cells, the
[Ca
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
Ca
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 Ca
The experiments shown in this article indicate
that this is indeed the case. Large, agonist-induced
[Ca
It can thus
be envisaged that to introduce the minimal artifactual quantitative or
qualitative changes in the Ca
Finally, cytAEQ
appears ideally suited for monitoring the Ca
In
conclusion, the cloning of the aequorin cDNA has largely extended the
use of this Ca
We thank G. Ronconi and M. Santato for technical
assistance; Y. Sakaki and S. Cotecchia for the aequorin and
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
] 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 Ca
probes 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.
binding, 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 Ca
indicator
(4, 5, 6, 7) .
indicators, 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 Ca
probe inside the cell
(1, 2, 3, 18, 19, 20, 21, 22) .
] 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 Ca
probes 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.
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) .
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 Ca
buffers were prepared by supplementing a
saline solution (130 mM KCl, 10 mM NaCl, 1
mM MgSO
, 0.5 mM
K
HPO
, 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 Ca
concentration of each buffer was calculated by a computer program
based on the Ca
affinity 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 Ca
indicator 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 Ca
buffers was injected in the
chamber. After 1 min, 2.5 µl of a 100 mM CaCl
solution 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 Ca
buffer, 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.
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 L
can 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 Ca
and detergents, or other
permeabilization procedures (see below), at the end of the experiment.
As aequorin is being consumed continuously, the value of
L
is not constant and decreases steadily during
the experiment. The value of L
to 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.
)
and [Ca
] has been modeled mathematically
(30) . The model preferred by the authors (model B) postulates
that each of the Ca
binding sites has two possible
states, T and R, and light is emitted when all of the sites are in the
R state. Ca
is 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
Ca
association constant; K
= [T]/[R]; and n, the number
of Ca
binding 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/ L
ratios. These data pairs, final pCa
and L/ L
ratios, 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/ L
corresponding 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
)
, and
K
, K
and 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 K
by 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 CaCl
solution
(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.
L
corresponds 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 Na
PO
, 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 Ca
buffering provided
by the fluorescent Ca
indicator.
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 Ca
homeostasis 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 G
protein 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 Ca
stores 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).
]
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 Ca
channels 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 Na
PO
, 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.''
-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 Ca
indicator 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 Ca
sensitivity of
native aequorin
(34, 35) and indicate that additions to
the amino terminus do not significantly modify the Ca
affinity 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
Ca
response curve of cytAEQ at physiological
conditions of pH, temperature, ionic strength, and
[Mg
].
]
estimates of
cytAEQ, both at rest and upon stimulation, closely match those of
fura-2, although, as expected from the steep Ca
response 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 Ca
probe in intact cells. Because of the high Ca
selectivity, 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 Ca
homeostasis 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) .
indicators, 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.
homeostasis (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.
]
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 Ca
buffering 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 Ca
is released, and in fact the
increase of the Ca
buffering capacity of the cell
invariably reduces the peak rise of
[Ca
]
(which largely
depends on the release of Ca
from intracellular
stores). Conversely, in the case of Ca
influx form
the external medium, a practically unlimited reservoir of
Ca
is available, and the steady-state
[Ca
]
level depends
solely on the relative rates of Ca
influx and
Ca
extrusion. Indeed, fura-2 loading causes no change
in the plateau phase of the
[Ca
]
rise, dependent
on the prolonged opening of plasma membrane channels.
dynamics, 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 Ca
signal. 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
Ca
buffering 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.
changes
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 Ca
signaling 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.
probe 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 Ca
indicator and, possibly, also to use this probe for studies
in situ.
], cytosolic
calcium concentration; PCR, polymerase chain reaction; cytAEQ,
cytosolic aequorin; PBS, phosphate-buffered saline; HEDTA,
N-hydroxyethylethylenediaminetriacetic acid.
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.