A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Article abstract of DOI:10.1002/anie.201902272

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura-2. Mt-fura-2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.

Full text of DOI:10.1002/anie.201902272

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Accepted Article
Title: A Synthetic Fluorescent Mitochondria-Targeted Sensor for
Ratiometric Imaging of Calcium in Live Cells
Authors: Diana Pendin, Rosa Norante, Andrea De Nadai, Gaia
Gherardi, Nicola Vajente, Emy Basso, Nina Kaludercic,
Cristina Mammucari, Cristina Paradisi, Tullio Pozzan, and
Andrea Mattarei
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201902272
Angew. Chem. 10.1002/ange.201902272
Link to VoR: http://dx.doi.org/10.1002/anie.201902272
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10.1002/anie.201902272
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COMMUNICATION
A Synthetic Fluorescent Mitochondria-Targeted Sensor for
Ratiometric Imaging of Calcium in Live Cells
Diana Pendin,*[a,b] Rosa Norante,[a] Andrea De Nadai,[c] Gaia Gherardi,[a] Nicola Vajente,[a] Emy
Basso,[a,b] Nina Kaludercic,[a,b] Cristina Mammucari,[a] Cristina Paradisi,[c] Tullio Pozzan,[a,b,d] and Andrea
Mattarei*[e]
Abstract: Ca2+ handling by mitochondria is crucial for cell life and
the direct measure of mitochondrial Ca2+ concentration in living cells
is of pivotal interest. Genetically-encoded indicators greatly
facilitated this task, however they require demanding delivery
procedures. On the other hand, existing mitochondria-targeted
synthetic Ca2+ indicators are plagued by several drawbacks, e.g.
non-specific localization, leakage, toxicity. Here we report the
synthesis and characterization of a new fluorescent Ca2+ sensor,
named mt-fura-2, obtained by coupling two triphenylphosphonium
cations to the molecular backbone of the ratiometric Ca2+ indicator
fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ~1.5 μM
in vitro. When loaded in different cell types as acetoxymethyl ester,
the probe shows proper mitochondrial localization and accurately
measures matrix [Ca2+] variations, proving its superiority over
available dyes. We describe the synthesis, characterization and
application of mt-fura-2 to cell types where the delivery of
genetically-encoded indicators is troublesome.
subcellular compartments.[10,11] However, experimental systems
exist where genetic manipulation is impractical, and the delivery
of GECIs is troublesome or plagued by cost- or safety-related
issues, requiring e.g. in situ electroporation or injection of viral
vectors.
Synthetic Ca2+ indicators represent a valid, easily deliverable
alternative. They comprise an organic fluorophore and
a
chelating domain, most often 1,2-bis(o-aminophenoxy)-ethane-
N,N,N′,N′-tetraacetic acid (BAPTA), with a high Ca2+ selectivity
over Mg2+.[12] Acetoxymethyl ester (AM) cell-permeant versions
of the indicators allow their loading into many cells at the same
time.[13,14] Problems associated with use of synthetic indicators,
i.e. heterogeneous loading, photobleaching and dye leakage are
minimized using dual-excitation (or -emission) wavelength
indicators, that yield calibrated [Ca2+] measurements through
ratiometry.[7]
The most used AM ester that accumulates in mitochondria is the
non-ratiometric sensor rhod-2-AM.[15,16] Despite its extensive use
by the scientific community, a number of studies demonstrated
that rhod-2 is plagued by serious drawbacks (e.g. non-specific
localization, leakage, toxicity),[9,17] and proved inadequate to
quantitatively monitor mitochondrial [Ca2+] ([Ca2+]m) changes.
Here we report the synthesis and characterization of a new
molecular tool based on the fura-2 dye[18] to monitor real-time
[Ca2+]m. To target the mitochondrial matrix, we used the
approach that exploits the negative potential across the
IMM[7,19,20] and developed a fura-2 analogue containing lipophilic
triphenylphosphonium (TPP) cations. TPP-containing molecules
have been reported to pass easily through the phospholipid
bilayers and selectively accumulate within the negatively
charged mitochondrial matrix in cultured cells[21,22] and in
vivo.[23,24]
Calcium (Ca2+) signalling is the trigger of essential physiological
processes.[1,2] Mitochondria, by taking up and releasing Ca2+ to
the cytosol, contribute to the shaping of cytosolic Ca2+ signals.
Matrix Ca2+, transported by the Mitochondrial Ca2+ Uniporter
(MCU), is essential for the regulation of specific mitochondrial
functions.[3–6] Several sensors have been generated to measure
Ca2+ inside mitochondria.[7–9] They are mostly genetically-
encoded Ca2+ indicators (GECIs) that can be targeted to specific
[a]
Dr. D. Pendin, Dr. R. Norante, A., Dr. G. Gherardi, N. Vajente, Dr. E.
Basso, Dr. N. Kaludercic, Dr. C. Mammucari, Prof. T. Pozzan
Department of Biomedical Sciences
University of Padua
Via U. Bassi 58/B 35131 Padua (IT)
In order to identify the best mitochondria-targeting strategy,
affording proper targeting and in situ functionality, we
synthesized and screened several fura-2 derivatives
incorporating one, two or three TPP groups linked to the probe
kernel at various positions (data not shown). We found that the
best performing molecule, hereafter named mt-fura-2 and
described in this work, contains two TPP lipophilic cations,
linked, respectively, one to the ethylenedioxy moiety of the
BAPTA chelating domain through an n-butyl spacer, the other to
the carboxylic function of the fluorophore via an N-propyl group
(Chart 1).
E-mail: diana.pendin@unipd.it
[b]
Dr. D. Pendin, Dr. E. Basso, Dr. N. Kaludercic, Prof. T. Pozzan
Neuroscience Institute, Padua Section
National Research Council
Via U. Bassi 58/B 35131 Padua (IT)
[c]
[d]
[e]
A. De Nadai, Prof. C. Paradisi
Department of Chemical Sciences
Via F. Marzolo 1 35131 Padua (IT)
Prof. T. Pozzan
Venetian Institute of Molecular Medicine
Via G. Orus 2 35131 Padua (IT)
Dr. A. Mattarei
Details on the 17-step synthesis, sketched in Scheme 1, and full
characterization of all intermediates and of the final product are
provided in the Supporting Information.
Department of Pharmaceutical and Pharmacological Sciences
University of Padua
Via F. Marzolo 5 35131 Padua (IT)
E-mail: andrea.mattarei@unipd.it
Supporting information for this article is given via a link at the end of
the document.
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Scheme 1. Synthesis of mt-fura-2 and mt-fura-2-AM Reagents and conditions: i) BnBr, K2CO3, DMF, 50°C, 16 h, 88% yield; ii) HNO3, AcOH, r.t., 16 h, 86%
yield; iii) TFA, DCM, r.t., 60 h, 96% yield; iv) DIPEA, DMF, 100°C, 72 h, 73% yield; v) DMAP, Py, TsCl, DCM, r.t., 12 h, 94% yield; vi) 5-methyl-2-nitrophenol,
KOH, DMF, 100°C, 4 h, 79% yield; vii) Pt/C, EtOAc, r.t., 75 min, 94% yield; viii) DIPEA, NaI, ethyl-2-bromoacetate, MeCN, reflux, 85 h, 96% yield; ix) POCl3,
DMF, 55°C, 16 h, 96% yield; x) Cyclohexene, Pd/C, THF, reflux, 5 h, 98% yield; xi) K2CO3, DMF, 85°C, 4 h, 63% yield; xii) Pd/C, H2, EtOAc, r.t., 2 h, 77% yield;
xiii) HATU, DMF, 3-chloroproylamine*HCl, r.t., 5 h, 51% yield; xiv) NaI, 2-butanone, 80°C, 16 h, 90% yield; xv) PPh3, neat, 90°C, 2.5 h, 96% yield; xvi) KOH,
H2O, MeOH, r.t., 4 h, 97% yield; xvii) DIPEA, bromomethyl acetate, DMF, r.t, 16 h, 58% yield.
to shorter wavelengths as [Ca2+] increases (Figure 1A), similarly
to the parent compound fura-2. However, emission spectra are
slightly shifted (+20 nm compared to fura-2). The dissociation
constant (Kd), calculated using Grynkiewicz’s equation,[18] is ~
1.5 µM (Figure 1B). Considering that physiological [Ca2+]m can
range between 100 nM and 10 µM, the Kd falls in the optimal
range to record mitochondrial Ca2+ increases upon stimulation,
but also to estimate resting [Ca2+]m. Conjugation with bulky and
positively charged TPP groups likely hinders the binding of Ca2+
due to steric effects and/or to interactions between the
negatively charged carboxylate groups and the TPP moiety,
resulting in an increase of the Kd, compared to the high affinity
parent compound fura-2 (Kd = 224 nM).[18]
Chart 1: Chemical structures of fura-2 and mt-fura-2.
Briefly, with reference to Scheme 1, the synthesis starts from p-
hydroquinone to build the BAPTA chelating moiety suitably
modified with a 4-chlorobutyl substituent on the ethylenedioxy
group (A10 in Scheme 1). Next, the furan-2-oxazole fluorophore
bearing a 3-chloropropyl amide group is introduced to achieve
intermediate A13, from which the final target, mt-fura-2, and its
membrane-permeant acetoxymethyl ester derivative, mt-fura-2-
AM, are readily accessed.
Table 1. Spectroscopic properties of mt-fura-2 free acid.
Absorbance max[a]
(nm)
Emission max[a]
(nm)
Ca2+-free
385
345
540
520
Ca2+-bound
Absorbance and fluorescence properties of the free acid form of
mt-fura-2 in the presence and absence of Ca2+ are reported in
Table 1 and Supplementary Figure 1A. Absorbance maxima
are in the near UV. The fluorescence excitation spectrum shifts
[a] Absorption maxima and emission maxima were recorded in a solution
containing, in mM: 130 KCl, 20 Hepes, 10 NaCl, 1 MgCl2, pH 7 at 37°C.
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Fluorescence spectra of a solution of mt-fura-2-AM (1 µM) at pH
8 in the presence of maximal or minimal Ca2+ have been
recorded over time to evaluate the hydrolysis of the AM
protecting groups at mitochondrial pH. An almost complete
chemical hydrolysis was observed after 20 hours
(Supplementary Figure 2A). However, unlike what observed for
fura-2,[25,18] the hydrolysis of AM groups did not cause an
increase in fluorescence intensity, nor in the fluorescence
quantum yield (ΦF) (Supplementary Figure 2A).
which was used in all the experiments reported. Also, these
results indicate that hydrolysis of mt-fura-2-AM in intact cells is
faster than in solution at pH 8 (see Supplementary Figure 2A
and Figure 2C, right), suggesting that hydrolysis is strongly
accelerated by some enzymatic activity within the mitochondrial
matrix.
To verify the accumulation within the mitochondrial matrix of the
membrane-permeant version of the probe, mt-fura-2-AM was
loaded into HeLa cells using an optimized version of the
classical protocol set for fura-2[26] (see Methods). Specific
mitochondria targeting was assessed at the confocal microscope
by evaluating colocalization of mt-fura-2 with a mitochondria-
targeted fluorescent protein (Figure 2A-B, Manders’ coefficient
= 0.90 ± 0.04). Partial retention in the cytosol was observed only
in a small fraction (8.0 ± 1.5 %) of cells. Permeabilization with
digitonin did not cause loss of mitochondrial staining nor
changes in cytosolic fluorescence in cells displaying proper
targeting (data not shown).
Figure 2: Loading of mt-fura-2-AM in HeLa cells. A) Confocal images of
HeLa cells transfected with a mitochondria-targeted RFP (red) and loaded with
mt-fura-2-AM (green) mt-fura-2. Scale bar 10 µm. B) Pixel intensity line-scan
referred to the dotted line in the merged image. C) mt-fura-2-AM was
incubated in solution at pH 8 (right) or loaded into intact HeLa cells (left). At
different times, the fluorescent signal was measured in the presence of 1 mM
Ca2+ or 500 µM EGTA and the dynamic range [R(max)/R (min)] was plotted
over time. D) Fluorescence imaging of mt-fura-2-AM loaded HeLa cells in the
presence of ionomycin (10 µM) and EGTA (500 µM) or CaCl2 (1 mM) as
indicated. Fluorescence emitted at 350 nm (left, red trace) or 400 nm (left, blue
trace) and the ratio between the two (right), normalized to their initial values,
were plotted. E) mt-fura-2-AM loaded HeLa cells were stimulated with 100 µM
histamine. Where indicated, 1 µM CCCP was present. Mean trace from a
single experiment is shown. Traces from single cells are reported in
Supplementary Figure 4.
Figure 2D shows that the mitochondria-trapped dye is sensitive
to changes in [Ca2+]. Mt-fura-2-loaded cells were alternatively
excited at 350 nm and 400 nm (See Methods and
Supplementary Figure 3) and the emission recorded during
perfusion of a saline containing the Ca2+-chelator EGTA in the
presence of the ionophore ionomycin. Upon switching to a saline
containing 1 mM CaCl2 the fluorescence elicited by excitation at
350 nm increased, while that recorded upon excitation at 400
nm decreased (Figure 2D, left panel) causing a marked
increase in the ratio R of the two signals (Figure 2D, right panel).
The mean dynamic range obtained using this protocol was about
3.
Figure 1. A) Excitation spectra of mt-fura-2 at the indicated [Ca2+]. Emission
was measured at 530 nm. B) Determination of the Kd value for Ca2+ binding to
mt-fura-2. See text for the definition of R. C) Effect of the indicated
concentration of Mg2+ on excitation spectra of mt-fura-2 D) Effect of pH on the
excitation spectrum of mt-fura-2, in the presence of saturating Ca2+ or of 500
µM EGTA. E) Effect of 700 µM H2O2 on the mt-fura-2 excitation spectra in the
presence of saturating Ca2+ or of 500 µM EGTA.
We thus verified that the internalized dye successfully converts
into the Ca2+-responsive tetra-acid form within the mitochondrial
matrix by recording emission spectra of mt-fura-2-loaded HeLa
cells (see Methods). As expected, the dynamic range increased
with incubation time (Figure 2C, left), reaching a plateau after
35 minutes of incubation. This represents the minimum
incubation time required for complete hydrolysis of AM groups,
Of note, the use of ionomycin may not guarantee full Ca2+
equilibration between the extracellular medium and the
mitochondrial matrix. Moreover, at high [Ca2+] we often observed
leakage of the probe, probably due to opening of mitochondrial
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Permeability Transition Pore (PTP). For these reasons, an
accurate determination of the apparent Kd in situ (that could
differ from the one calculated in vitro)[27] was hard to obtain. We
attempted an approximate estimate by passive equilibration of
the mitochondrial matrix with a saline containing known [Ca2+] in
the presence of the uncoupling agent CCCP (see Methods for
details). The resulting Kd in situ was estimated to be around 6
µM.
The ability of the probe to record a transient [Ca2+]m increase
was assessed by addition of IP3-generating stimulus histamine
to the cells. As predicted, R transiently increased (Figure 2E).
The exclusive mitochondrial localization of the probe was further
confirmed by the fact that R350/400 did not change in the presence
of CCCP (Figure 2E). Next, we verified whether the new probe
suffers from the problems reported for rhod-2. Supplementary
Figure 5 shows confocal images of HeLa cells transfected with
mitochondria-targeted GFP and then incubated with rhod-2,
where a cytosolic staining is clearly appreciable. Accordingly,
the mean ratio between the fluorescence intensity recorded in
the mitochondria and in the nucleus is 2.0 with rhod-2 and 8.4
with mt-fura-2 (Figure 2A), i.e., 4.2-fold higher with the new
probe. Two typical problems related to the intensometric nature
of rhod-2, that are successfully overcome using ratiometric mt-
fura-2, are presented in Figure 3A-D.
Figure 3: A-B) HeLa cells loaded with mt-fura-2-AM (A) or rhod-2-AM (B)
imaged immediately after washing or after 15 minutes of illumination (150 ms
every 5 s). Scale bar 10 µm. C-D) Fluorescence traces over time (150 ms
exposure every 5 s) of cells loaded with mt-fura-2-AM (C) or rhod-2 (D).
Intentional changes in the focal plane, causing a decrease in fluorescence
visible in single wavelength recordings (C, left plot), are marked with arrows.
E) Effect of two subsequent applications of 100 µM histamine to HeLa cells
loaded with mt-fura-2-AM. Dotted line represents a 10 min pause in acquisition.
F) HeLa cells loaded with mt-fura-2-AM were incubated for 3 hours in EC
solution in the presence or absence of 0.8 µM Cyclosporin A. Fluorescence
intensity (AU) was measured upon excitation at 400 nm. Data are presented
as mean ± SEM; n ≥ 5; ***p < 0.001.
Cells loaded with rhod-2 were illuminated every 5 seconds for 15
minutes, resulting in
a significant drop (about 60%) of
fluorescence (Figure 3B,D) probably due to photobleaching of
the probe. This flaw is amended by the ratiometric properties of
mt-fura-2: not only the probe is less bleached upon exposure to
light (Figure 3A,C, left), but also the decrease in fluorescence
recorded at the two excitation wavelengths is corrected by the
calculation of R (Figure 3C, right). Moreover, when mt-fura-2-
labelled cells were on purpose defocused to mimic a movement
artifact, the change in fluorescence, mimicking a variation in
[Ca2+] in single wavelengths plots, has no impact on R (Figure
3C).
Alvarez et al. have shown the inability of rhod-2 to follow
adequately [Ca2+]m dynamics during repetitive stimulation,[17]
likely due to photobleaching and/or exit of the probe from
mitochondria. On the contrary, two subsequent IP3-generating
stimuli were adequately recorded by mt-fura-2 (Figure 3E).
Notably, the second histamine-induced peak is decreased
compared to the first. This is probably ascribable to a partial de-
sensitization of plasma membrane histamine receptor, indeed a
similar decrease is observed also in the cytosol
(Supplementary Figure 6). Altogether, the results obtained
clearly confirm the advantages of using mt-fura-2 compared to
the only other probe available for mitochondrial Ca2+
measurements.
Potential mitochondria toxicity induced by the mt-fura-2-AM
internalization and/or hydrolysis has been evaluated by
measuring oxygen consumption rate (OCR). Cells loaded with
mt-fura-2 did not display significant differences in basal nor
maximal OCR, when compared with control, unloaded cells
(Figure 4A-B).
Figure 4: A) Oxygen consumption rate (OCR) was measured in HeLa cells
loaded with mt-fura-2-AM (2 μM) and in control cells. Maximal respiration was
induced by addition of 0.3 μM CCCP. B) Quantification of basal and maximal
respiration. Data are presented as mean ± SEM; n ≥ 60.
Despite the adequate retention of the probe for the time course
of standard experiments, we noticed that prolonged washing
resulted in a significant loss of mitochondrial fluorescence,
possibly due to flickering of the PTP.[28] Indeed, this effect was
largely prevented by pre-incubation with cyclosporin A (Figure
3F).
The probe was then tested in experimental systems where the
delivery of GECIs via transfection is troublesome or plagued by
cost- or safety-related issues. Figure 5A shows cardiomyocytes
isolated from adult mice, loaded with mt-fura-2 and imaged at
the confocal microscope. Proper mitochondrial localization was
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10.1002/anie.201902272
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COMMUNICATION
confirmed by concomitant loading with TMRM. Transient
mitochondrial Ca2+ increase induced by stimulation with KCl was
adequately mirrored by mt-fura-2 (Figure 5B).
Cultured astrocytes isolated from neonatal mice cortex were
loaded with mt-fura-2 (Figure 5C) and stimulated with ATP. As
expected, mt-fura-2 was able to nicely report the transient
increase in mitochondrial Ca2+ (Figure 5D). Finally, freshly
isolated flexor digitorum brevis (FDB) muscle fibres were loaded
the resting levels, correctly reporting the decrease in basal
[Ca2+]m when MCU is downregulated. The new probe has largely
retained the spectroscopic properties of the parent compound
fura-2, including UV excitation, which is known to result in
cellular toxicity, particularly in long imaging experiments. The
strategy we developed, however, can be in principle applied to
other Ca2+ sensors, paving the way for the development of a
new class of mitochondria-targeted Ca2+ probes.
with mt-fura-2 and stimulated with caffeine.
A
robust
mitochondrial Ca2+ uptake was measured by mt-fura-2 (Figure
5E). Notably, the uptake was significantly reduced in fibres
where MCU[29] was downregulated (Figure 5F). Moreover, in this
latter system mitochondrial resting [Ca2+] appeared significantly
lower, compared to controls, consistently with previous results[30]
(Figure 5G).
Acknowledgements
We thank Mario Zoratti and Cristina Fasolato for helpful advice
and discussion; Elena Gurrieri for help with fluorimeter
measurements. This work is funded by the University of Padua
(STARS Grants programme “CHEMPROCALIM” to A.M.), MIUR
(PRIN to T.P.), the Italian Ministry of Education (Euro
Bioimaging Project to T.P.), Fondazione Cassa di Risparmio di
Padova e Rovigo (CARIPARO) Foundation (to T.P. and Starting
Grant 2015 to D.P.), Consiglio Nazionale delle Ricerche (CNR)
(Special Project Aging to T.P.) and Telethon Foundation (to
T.P.).
Keywords: Fluorescent probes • Bioimaging • Calcium •
Mitochondria • Fura
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10.1002/anie.201902272
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
mt-fura-2 is a selective mitochondria-
targeted ratiometric fluorescent
Diana Pendin,* Rosa Norante, Andrea
De Nadai, Gaia Gherardi, Nicola
chemical Ca2+ sensor, obtained by
coupling two triphenylphosphonium
cations to Ca2+ probe fura-2. When
loaded in different cell types as
acetoxymethyl ester, the probe shows
proper mitochondrial localization and
accurately measures matrix [Ca2+]
variations, proving its superiority over
available non-ratiometric dyes.
Vajente, Emy Basso, Nina Kaludercic,
Cristina Mammucari, Cristina Paradisi,
Tullio Pozzan and Andrea Mattarei*
((Insert TOC Graphic here))
Page No. – Page No.
A Synthetic Fluorescent
Mitochondria-Targeted Sensor for
Ratiometric Imaging of Calcium in
Live Cells
This article is protected by copyright. All rights reserved.