Red fluorescent probe for monitoring the dynamics of cytoplasmic calcium ions

Article abstract of DOI:10.1002/anie.201210279

We see red (and yellow and green): Probe 1 was developed for the visualization of cytoplasmic Ca²⁺, a pivotal second messenger in many biological responses. The new probe is suitable for multicolor imaging for the simultaneous detection of metal ions or proteins and is superior to the existing red fluorescent probe Rhod-2 for the monitoring of cytoplasmic Ca²⁺ oscillation in cultured cells (see fluorescence images of cells with 1 (top) and Rhod-2 (bottom)).

Full text of DOI:10.1002/anie.201210279

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Angewandte
Communications
DOI: 10.1002/anie.201210279
Molecular Probes
Red Fluorescent Probe for Monitoring the Dynamics of Cytoplasmic
Calcium Ions**
Takahiro Egawa, Kazuhisa Hirabayashi, Yuichiro Koide, Chiaki Kobayashi, Naoya Takahashi,
Tomoko Mineno, Takuya Terai, Tasuku Ueno, Toru Komatsu, Yuji Ikegaya, Norio Matsuki,
Tetsuo Nagano, and Kenjiro Hanaoka*
The development of sophisticated fluorescent probes has
contributed to the elucidation of the molecular mechanisms
of many complex biological phenomena.[1–4] In particular,
fluorescence imaging of the calcium ion (Ca2+) has become an
essential technique for the investigation of signaling pathways
involving Ca2+ as a second messenger. For example, changes
in the intracellular Ca2+ concentration have been found to be
related to physiological responses in obesity, as well as
immune responses and pathological responses in Alzheimerꢀs
disease.[5–10] Because Ca2+ signaling is involved in so many
biological phenomena,[11,12] it is expected that the simulta-
neous visualization of Ca2+ and other biomolecules, that is,
multicolor imaging, would be particularly informative. For
this purpose, we require a fluorescent probe for Ca2+ that
operates in a different color window from that of probes for
other molecules.
fluorescence (ca. 527 nm).[15–17] There are also some red-
emitting fluorescent probes for Ca2+, such as Rhod-2 (ca.
576 nm), which is based on the rhodamine scaffold.[15] These
red-emitting fluorescent probes for Ca2+, including Rhod-2,
are also widely used for biological studies; however, the
cationic nature of the rhodamine scaffold generally causes
Rhod-2 AM to localize into mitochondria.[18] Although this
behavior is useful for monitoring the Ca2+ dynamics of
mitochondria, the visualization of cytoplasmic Ca2+ is much
more important for research on Ca2+ signaling. The influx of
Ca2+ into the cytoplasm from the extracellular environment
and/or from intracellular stores (including the endoplasmic
reticulum) triggers numerous cellular responses mediated by
the interaction of Ca2+ with various Ca2+-binding proteins,
such as calmodulin and troponin C.[11,12] Fura Red is a repre-
sentative near-infrared fluorescent probe for Ca2+ that is
often used in biological research. However, it has extremely
low fluorescence quantum efficiency (Ffl ꢀ 0.013).[19] Accord-
ingly, the fluorescence signal is very small unless a high
concentration of Fura Red or a high-powered laser is used.
However, the use of a high dye concentration has a buffering
effect on Ca2+, whereas the use of a high laser power causes
rapid photobleaching of the dye and phototoxicity to the cells.
Thus, a novel fluorescent probe for cytoplasmic Ca2+ with
strong emission in the long-wavelength region would be
extremely useful, especially for multicolor imaging. In the
present study, we designed and synthesized a novel and
practical red-fluorescence-emitting probe suitable for mon-
itoring cytoplasmic Ca2+ and confirmed its usefulness for the
visualization of stimulus-induced Ca2+ oscillation in HeLa
cells.
As a fluorophore that emits in the red region, we chose
TokyoMagenta (TM). The absorption and fluorescence wave-
lengths of this fluorescein analogue are 90 nm longer than
those of fluorescein.[20] TM was also expected to retain the
advantages of the fluorescein scaffold, including cytoplasmic
localization. For the development of the red fluorescent
probe, we chose a combination of 2-Me-substituted TM as the
fluorescent moiety and 1,2-bis(o-aminophenoxy)ethane-
N,N,N’,N’-tetraacetic acid (BAPTA) as a specific chelator
for Ca2+, and synthesized CaTM-1 (Figure 1; see also
Scheme S1 in the Supporting Information).
The fluorescence-activation ratio of CaTM-1 in the
presence/absence of Ca2+ is 5.6:1 (Figure 2a,b, Table 1). To
further improve this ratio, we decided on the strategy of
decreasing the energy of the highest occupied molecular
orbital (HOMO) of the fluorophore to obtain a high level of
Fluorescent Ca2+ sensors can be categorized into two main
classes: those based on genetically encoded fluorescent
proteins[13,14] and those based on fluorescent small organic
molecules.[5] Although both types of sensors have certain
advantages and drawbacks, small-molecule-based probes
have the particular advantage that their AM ester form
(cell-permeable acetoxymethyl ester derivative) can be read-
ily bulk loaded into live cells with no need for transfection.
Most currently used small-molecular fluorescent probes for
Ca2+ are fluorescein-based, such as Fluo-3, Fluo-4, Calcium
Green-1, and Oregon Green 488 BAPTA-1, and emit green
[*] T. Egawa, K. Hirabayashi, Dr. Y. Koide, C. Kobayashi,
Dr. N. Takahashi, Dr. T. Terai, Dr. T. Ueno, Dr. T. Komatsu,
Dr. Y. Ikegaya, Prof. N. Matsuki, Prof. T. Nagano, Dr. K. Hanaoka
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
E-mail: khanaoka@mol.f.u-tokyo.ac.jp
Dr. T. Mineno
Faculty of Pharmacy, Takasaki University of Health and Welfare
60 Nakaorui, Takasaki-shi, Gunma 370-0033 (Japan)
[**] This research was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology of Japan (Specially
Promoted Research Grant No. 22000006 to T.N.), SENTAN, the JST
(grant to K.H.), and the Funding Program for Next Generation
World-Leading Researchers (LS023 to Y.I.). K.H. was also supported
by grants from the Inoue Foundation for Science, the Takeda
Science Foundation, the Tokyo Biochemical Research Foundation,
and the Astellas Foundation for Research on Metabolic Disorders.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201210279.
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Figure S1 in the Supporting Information). The HOMO
energy level of DCTM was calculated to be lower than that
of TM (see Table S1 in the Supporting Information). We then
developed a novel Ca2+ probe, CaTM-2, based on DCTM
(Figure 1; see also Scheme S1 in the Supporting Information).
As we had hoped, the activation ratio of CaTM-2 was
considerably improved (16:1) with respect to that of CaTM-1,
presumably owing to stabilization of the HOMO energy level
of the fluorophore moiety (Figure 2c,d, Table 1).
The chlorination of the fluorophore was also advanta-
geous in another respect. As we have previously reported, TM
shows pH dependency: the absorption wavelength is mark-
edly blue-shifted under acidic conditions (see Figures S2–S4
and Table S2 in the Supporting Information),[20] and CaTM-
1 showed similar behavior (Figure 3). This property can
Figure 1. Left: Chemical structures of the red fluorescent Ca2+ probes
CaTM-1, CaTM-2, and CaTM-2 AM (a cell-permeable derivative of
CaTM-2). Right: Photographs of an aqueous solution of CaTM-2 in the
absence of Ca2+ and in the presence of free Ca2+ ions (39 mm) under
UV irradiation (365 nm).
Figure 3. pH dependency of CaTM-1 and CaTM-2. a) pH-dependent
equilibrium of CaTM-1 and CaTM-2 in an aqueous buffer. b) Absorp-
tion spectra of CaTM-1 (red) and CaTM-2 (blue) in acidic (pH 3) and
basic (pH 9) sodium phosphate buffer containing ethylenediaminete-
traacetic acid (EDTA; 5 mm). For convenience for comparison, the
relative absorption is shown, that is, the maximum absorption of each
anion form was defined as 1.0. c) Curve fittings of pH-dependent
changes in the normalized absorbance of CaTM-1 (red) and CaTM-2
(blue) at the absorption maximum for each anion form (right-hand
structure in Figure 3a; 585 nm for CaTM-1 and 597 nm for CaTM-2) in
sodium phosphate buffer containing EDTA (5 mm).
Figure 2. a,b) Ca2+-dependent absorption (a) and emission (b) spectra
of CaTM-1 (1 mm) in the presence of free Ca2+ ions at various
concentrations (0, 0.017, 0.038, 0.065, 0.100, 0.150, 0.225, 0.351,
0.602, 1.35, 39 mm) in 3-(N-morpholino)propanesulfonic acid (MOPS)
buffer (30 mm) containing KCl (100 mm) and ethyleneglycol tetraacetic
acid (EGTA; 10 mm) at pH 7.2 and 228C. The excitation wavelength
was 550 nm. The relative absorption and relative fluorescence intensity
are shown. c,d) Ca2+-dependent absorption (c) and emission (d)
spectra of CaTM-2 (1 mm) under the same conditions as in (a,b).
Table 1: Photophysical properties of CaTM-1 and CaTM-2.[a]
reduce the fluorescence signal under physiological conditions
(pH 7.4), and a sufficiently low pKa value of the probe is
required for biological applications. Fortunately, the
pKa value of CaTM-2 was greatly shifted into the acidic
region relative to that of CaTM-1 (Figure 3), and was
sufficiently low (pKa = 5.1) for practical use. This pKa value
is derived from that of the fluorophore itself (Figure 3a), that
is, the lower pKa value of CaTM-2 is due to the electron-
withdrawing character of chlorine (see Figures S2–S5 and
Table S2 in the Supporting Information). We next examined
the suitability of CaTM-2 as a red fluorescent probe for
monitoring cytoplasmic Ca2+ in cells.
[Ca2+]=0 mm
[Ca2+]=39 mm
labs
[nm]
lfl
Ffl
labs
[nm]
lfl
Ffl
Kd
[mm]
[nm]
[nm]
CaTM-1
CaTM-2
585
597
603
609
0.066
0.024
585
597
603
609
0.37
0.39
0.38
0.20
[a] Measurements were made in a buffer under the conditions described
in Figure 2 in the absence and presence of free Ca2+ ions.
quenching by photoinduced electron transfer in the absence
of Ca2+.[21] With this aim, we introduced chlorine into the
fluorophore to produce dichloro-TokyoMagenta (DCTM; see
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For cellular applications, we synthesized CaTM-2 AM, an
AM ester form of CaTM-2 (Figure 1; see also Scheme S1 in
the Supporting Information). Because of the hydrophilicity of
the BAPTA moiety, Ca2+ indicators, including CaTM-2,
generally cannot pass through cell membranes without the
use of special procedures, such as electroporation.[22] In
contrast, AM ester forms of Ca2+ probes can readily enter
cells, where they are enzymatically cleaved by intracellular
esterase to afford the Ca2+-sensitive, cell-impermeable form.
To examine the potential of CaTM-2 for multicolor
imaging, we loaded HeLa cells expressing cyan fluorescent
protein (CFP; localized to the nucleus) and yellow fluorescent
protein (YFP; localized to the Golgi apparatus) with CaTM-
2 AM (Figure 4). The fluorescence of CaTM-2 in the red
and enabled the successful visualization of changes in the
cytoplasmic Ca2+ concentration (Figure 5d; see Supporting
movie 1). Ca2+ spikes did not occur in all cells, and the
oscillation behavior depended on the cell. We also performed
the equivalent experiment with Rhod-2 AM (Figure 5e–h). In
the histamine-stimulated cells, most of the areas that showed
an increase in fluorescence were mitochondria (Figure 5g), in
agreement with previous reports.[24–27] Thus, Rhod-2 AM
mainly monitored the Ca2+ concentration in mitochondria,
which exhibit a slower kinetic Ca2+ transient relative to that in
the cytosol (Figure 5h; see also Supporting movie 2).[25] We
examined the localization of these probes in other cell lines
(COS7, A549, CHO-k1, and HEK293) and observed no
distinctive compartmentalization of CaTM-2 AM inside these
cells (see Figure S6 in the Supporting Information). On the
other hand, Rhod-2 AM was mostly localized in compart-
ments that appeared to be mitochondria, in accordance with
previous reports (see Figures S6–S8 in the Supporting Infor-
mation).[24–27]
We also compared the usefulness of CaTM-2 AM and
Fura Red as fluorescence imaging agents. CaTM-2 AM could
clearly detect ATP-induced Ca2+ oscillation and ionomycin-
induced Ca2+ influx in HeLa cells (see Figure S9 in the
Supporting Information; ATP = adenosine triphosphate).
Fura Red could also be used to monitor these events, but
high-power laser excitation was needed to obtain a sufficient
fluorescence signal, probably because of the very low
fluorescence quantum efficiency of Fura Red, and the
fluorescence signal was still a little noisy (see Figure S10 in
the Supporting Information). Furthermore, CaTM-2 is
expected to be superior to Fura Red for Ca2+ imaging in
biosamples, such as tissues, because the excitation and
emission wavelengths of CaTM-2 both lie in the tissue-
penetrating long-wavelength region, whereas Fura Red has
a much shorter excitation wavelength.[28] However, Fura Red
has the strong point that it can be used for ratiometric
imaging, unlike CaTM-2. We utilized a commercially avail-
able dye-dispersing agent, Pluronic F-127, to assist in loading
the fluorescent probe into live cells (see Figures S9 and S10).
Figure 4. Triple-color imaging of CFP, YFP, and CaTM-2 in HeLa cells.
a–c) CFP–nucleus (a, cyan channel) and YFP–Golgi (b, yellow channel)
transfected Hela cells were loaded with CaTM-2 AM (c, red channel).
d) Triple-color merged image. Scale bar: 20 mm.
region was clearly separable from the signals of CFP and YFP.
Thus, the multicolor imaging of cytoplasmic Ca2+ simulta-
neously with plural biomolecules can be carried out by
utilizing CaTM-2 AM together with fluorophores whose
fluorescence wavelengths are in the yellow or shorter-wave-
length region (including well-established fluorescent protein-
based sensors).[13,23]
Next, we examined whether CaTM-2 can detect changes
in intracellular cytoplasmic Ca2+ concentration. HeLa cells
were loaded with CaTM-2 AM and then stimulated with
histamine to induce Ca2+ oscillation (Figure 5a–d).[24] As
expected, CaTM-2 diffused uniformly throughout the cytosol,
Figure 5. Visualization of histamine-induced calcium oscillations in HeLa cells with CaTM-2 AM and Rhod-2 AM. HeLa cells loaded with CaTM-2 AM (3 mm;
a–d) or Rhod-2 AM (3 mm; e–h) were stimulated with histamine (1 mm). a–c) Fluorescence images of CaTM-2. Images (b) and (c) were taken at the time
points indicated with arrowheads in (d). e–g) Fluorescence images of Rhod-2. Images (f) and (g) were taken at the time points indicated with arrowheads
in (h). d,h) Fluorescence changes in regions of interest of individual cells numbered 1–7 in (a) and (e) are shown in (d) and (h), respectively. Scale bars:
30 mm. See also Supporting movies 1 and 2.
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The use of the dispersant greatly enhanced the emission
signal. Thus, the loading of CaTM-2 AM with a dispersing
agent is effective in producing a large fluorescence signal,
although it may have some influence on cellular homeostasis.
For the broader biological application of Ca2+ indicators
for multicolor imaging and in vivo imaging, some red to near-
infrared fluorescent probes for Ca2+, CaSiR-1,[29] KFCA,[30]
and Calcium Rubies[31] were recently developed. CaSiR-1 and
its AM ester form are especially useful for neuronal Ca2+
imaging, but because of its localization in lysosomes in some
types of cultured cells, CaSiR-1 AM will sometimes be
unsuitable for the detailed analysis of signaling pathways
inside cells (see Figures S11–S15 in the Supporting Informa-
tion). Two other probes were also used successfully to monitor
cytoplasmic Ca2+; however, the AM ester forms of these
probes were not synthesized. They were introduced into cells
by bead loading[30] or through unique uptake into astro-
cytes.[31] Unlike these probes, CaTM-2 could be used to
monitor cytoplasmic Ca2+ in cultured cells by using the
AM ester loading method.
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Received: December 25, 2012
Revised: January 26, 2013
Published online: February 25, 2013
Keywords: bioimaging · calcium · cell signaling · fluorescence ·
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imaging agents
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