Intracellular click reaction with a fluorescent chemical Ca2+ indicator to prolong its cytosolic retention

Article abstract of DOI:10.1039/c3cc42489h

The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca²⁺ indicator in the cytosol. Specifically, a novel clickable Ca²⁺ indicator "N₃-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca²⁺ oscillation was observed for an extended period of time.

Full text of DOI:10.1039/c3cc42489h

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Intracellular click reaction with a fluorescent chemical
Ca2+ indicator to prolong its cytosolic retention†
Yoshiaki Takei,a Atsushi Murata,a Kento Yamagishi,a Satoshi Arai,b
Hideki Nakamura,a Takafumi Inouea and Shinji Takeoka*a
Cite this: Chem. Commun., 2013,
49, 7313
Received 6th April 2013,
Accepted 16th May 2013
DOI: 10.1039/c3cc42489h
www.rsc.org/chemcomm
The powerful strategy of ‘‘intracellular click reaction’’ was used to retain a membrane to dextran-conjugated dyes the troublesome micro-
chemical Ca2+ indicator in the cytosol. Specifically, a novel clickable Ca2+ injection technique is required for cell loading.
indicator ‘‘N3-fura-2 AM’’ was coupled with dibenzylcyclooctyl-modified
Here, we propose a convenient strategy to prolong the cytosolic
biomacromolecules via copper-free click reaction in living cells and Ca2+ retention by utilizing ‘‘intracellular click reaction’’ with cytosolic
oscillation was observed for an extended period of time.
biomacromolecules such as proteins. We synthesized a fura-2
derivative ‘‘N3-fura-2 AM’’ as a clickable Ca2+ indicator as shown
Changes in the intracellular concentration of calcium (Ca2+) is an in Fig. 1. Fura-2 AM is a ratiometric chemical Ca2+ indicator that
indispensable signal involved in the regulation of activities1 such as allows the ratiometric measurement of Ca2+ concentration by dual
neural transmission,2,3 muscle contraction4,5 and fertilization.6 The excitation (lEx, 340 nm/380 nm; lEm, 510 nm). N3-fura-2 AM is
recent development of Ca2+ indicators, such as small chemical capable of coupling with molecules containing an alkyne moiety
fluorescent dyes or genetically encoded fluorescent proteins, has via azide–alkyne 1,3-dipole cycloaddition (Huisgen reaction), which
allowed various biological events related to Ca2+ signaling to be is an example of a ‘‘click reaction’’.18 N3-fura-2 AM also has an
visualized.7 Although genetic approaches based on Ca2+-sensitive acetoxymethyl (AM) ester to improve its membrane permeability.19
fluorescent proteins (e.g. cameleon,8 G-CaMP9) have proved to be Compared with other intracellular labeling strategies, the click
powerful tools for fluorescent microscopic observations in living reaction has an advantage in terms of being rapid and bioortho-
animals, tissues or primary cultured cells, effective expression of gonal.20 More recent advances in copper-free click chemistry using
these sensor proteins requires laborious steps such as production a cyclooctyne group allow us to perform click chemistry in the
and maintenance of transgenic animals or viral vector preparation.10 absence of copper catalysts, which are cytotoxic.21 Thus we
By contrast, small chemical Ca2+ indicators (e.g. fura-2,11 fluo-3, employed a copper-free click reaction to conjugate our Ca2+
rhod-212) are easy to handle for staining tissue as well as cultured indicator with biomacromolecules in the cytosol. This strategy
cells. However, these chemical Ca2+ indicators generally have too circumvents the problem of leakage of the Ca2+-indicator from
short cytosolic retention time for useful microscopic observations.13 the cell and allows the indicator to retain its function for an
This problem arises because these indicators leak from the cell or extended period of time.
accumulate in organelles due to the action of the anion transpor-
We designed N3-fura-2 AM bearing an azide moiety at the
ters.14,15 To date, two strategies to prolong the cytosolic retention of terminus of the alkyl chain so that the clickable site is maintained
Ca2+ indicators have been reported. One strategy involves modifica- at a distance from the chelating ligand that binds Ca2+. N3-fura-2
tion of the dye with cationic charges, which allow it to evade the and N3-fura-2 AM were synthesized from p-hydroquinone through
activity of the anionic transporter. One such example is the commer- 14 and 15 steps (see ESI†), respectively.
cially available cationic Ca2+ indicator fura-PE3,16 although it still
tends to accumulate in organelles during incubation. The second
strategy involves conjugation of the dyes to macromolecules such as
dextran.17 However, due to the impermeability of the plasma
a Department of Life Science & Medical Bioscience, Graduate School of Advanced
Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku,
02C227, TWIns, Tokyo, 162-8480, Japan. E-mail: takeoka@waseda.jp
b Waseda Bioscience Research Institute in Singapore, Waseda University,
11 Biopolis Way, #05-01/02, Helios, Singapore, 138667, Singapore
Fig. 1 Chemical structure of N3-fura-2 AM and schematic illustration of the copper-
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc42489h
free click reaction with dibenzylcyclooctyl (DBCO)-modified biomolecules.
c
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The critical step in the synthetic procedure was deprotection of
the benzyl group of the precursor. Deprotection by catalytic
reduction was deemed unsuitable because the azide group was
likely to be reduced. Thus, we chose a deprotection method using
MgBr2, which is based on the selective cleavage of an aromatic
benzyl group at the ortho position to a carbonyl group.22
The Ca2+ sensitive excitation spectrum of N3-fura-2 (Fig. 2b) was
similar to that of fura-2 (Fig. S2, ESI†). The affinity of N3-fura-2 for
Ca2+ (Kd = 304 nM at pH 7.2, 28 1C) was relatively low by
comparison to that of conventional fura-2 (Kd = 125 nM under
the same conditions). We assume that the alkyl chain attachment
might affect the flexible region of the Ca2+ chelating ligand.23
In order to evaluate the clickability of N3-fura-2 (AM), we prepared
bovine serum albumin (BSA) modified with dibenzylcyclooctyl (DBCO)
as a copper-free click reagent.24 DBCO-modified BSA was prepared
using DBCO succinimidyl ester, which was non-selectively conjugated
to amine groups of BSA. DBCO-modified BSA and N3-fura-2 AM
Fig. 3 Confirmation of intracellular click reaction of N3-fura-2 with DBCO-
modified biomacromolecules. Fluorescence images of HeLa cells ((a and b) N3-
fura-2 and (c and d) fura-2 at 5 min and 1 hour after washing, respectively). The
scale bar is 20 mm. (e) SDS-PAGE analysis of the lysate after intracellular click
reaction with N3-fura-2. The fura-2 dye was added to the cells after DBCO
modification treatment. The gel was (left) stained with CBB and (right) irradiated
with UV at 365 nm.
(10 mM) were mixed in an aqueous buffer solution. We checked how very weak compared with that of the cytosol. In contrast, for fura-2
the click reaction proceeded in the sample tube by SDS-PAGE analysis the difference in the intensities between the cytosol and the
(Fig. 2a). After mixing DBCO-modified BSA and fura-2 AM under the nucleus was very small (the detailed description of the intracellular
same conditions, fluorescence was barely observable at the band of localization of N3-fura-2 is provided in ESI†). These observations
BSA stained with Coomassie Brilliant Blue. This result suggests that imply that N3-fura-2 was conjugated with cytosolic or membrane
the fluorescence labeling of BSA was caused by coupling of N3-fura-2 proteins, thereby preventing the fluorescent dye from penetrating
AM and DBCO-modified BSA via the copper-free click reaction. There through the nuclear pores. The SDS-PAGE analysis of lysates
was no non-specific binding of fura-2 to BSA.
from N3-fura-2 stained cells showed many fluorescent bands in
We further investigated the influence of the BSA conjugation on the entire lane and indicated the conjugation between N3-fura-2
the Ca2+-indicating function of the fura-2 moiety by comparison to and various intracellular proteins (Fig. 3e). Therefore, the
that of free N3-fura-2. As shown in Fig. 2c, both the excitation fluorescence intensities in the case of N3-fura-2 should have
spectrum and the affinity after click reaction with DBCO-modified originated from the intracellular click reaction of N3-fura-2 with
BSA (Kd = 298 nM) were almost identical to those before the intracellular DBCO-modified proteins.
reaction (Kd = 304 nM). This finding suggests that macromolecules
Finally, we evaluated the cytosolic retention time of N3-fura-2
such as BSA conjugated by click reaction do not affect the proper- by comparison to those of fura-2 and a cationic long-term Ca2+
ties of the fluorophore and the environment surrounding the indicator (fura-PE3). Fig. 4a and b shows a series of fluorescence
chelating structure of fura-2.
images of Ca2+ indicator (N3-fura-2, fura-2) loaded HeLa cells
Next, we performed the intracellular click reaction in HeLa cells (images of fura-PE3 are shown in Fig. S6, ESI†). As expected,
by using N3-fura-2 AM. Firstly, the intracellular environment inside N3-fura-2 after intracellular click reaction remained in the cytosol for
HeLa cells was non-specifically modified with DBCO succinimidyl a longer period of time than fura-2 and fura-PE3. Quantitative
ester. N3-fura-2 AM or fura-2 AM was then applied to DBCO modified analyses of the cytosolic retention of the fluorescence signal
HeLa cells using the same procedure (1 mM loading solution was indicated a significant difference between conventional Ca2+ indica-
incubated with cells for 30 min at 37 1C). The fluorescence micro- tors and N3-fura-2 (p value = 0.003 between N3-fura-2 and fura-PE3 at
scopic images of N3-fura-2 stained cells at 5 min after washing 180 min after loading) (Fig. 4c). The time taken for the fluorescence
(Fig. 3a) showed the fluorescence intensity of the nucleus was to decrease to half the initial level for fura-2, fura-PE3 and N3-fura-2
was 35 min, 63 min and 185 min, respectively. For short term
measurements, leakage of dye into the medium might cause
baseline drift, regardless of whether or not ratiometric methods
are employed. Indeed, during microscopic observation without
stimulation of cells, the apparent Ca2+ dependent fluorescence
ratio of fura-2 did drift (Fig. 4d). By contrast, N3-fura-2 after
conjugation with intracellular macromolecules did not show
such a drift, thereby enhancing the accuracy of the measure-
ments. In addition, to determine the duration of effective dye
retention, we checked whether indicators can work with ligand
(histamine) mediated Ca2+ oscillation at 5 min, 3 hours and
Fig. 2 Identification of N3-fura-2 coupled DBCO-modified BSA. (a) SDS-PAGE
analysis of DBCO-modified BSA coupled with N3-fura-2 and the mixture of DBCO-
6 hours after dye loading. At 5 min after loading, the fluorescence
modified BSA with fura-2. The gel was imaged whilst being (top) stained with
ratio of both fura-2 and N3-fura-2 indicated similar transient Ca2+
Coomassie Brilliant Blue (CBB) and (bottom) irradiated with UV at 365 nm.
Excitation spectra of N3-fura-2 at different Ca2+ concentrations (b) before and
(c) after BSA conjugation.
elevation, showing little influence of DBCO and N3-fura-2 conjuga-
tion on the Ca2+ oscillation and its detection. However, in the case
c
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concentrations of Ca2+ during various biological processes, including
the circadian rhythm25 and embryonic development,26 as well as
in vivo Ca2+ imaging. Even during short-term measurements of
intracellular Ca2+ levels, leakage and compartmentalization of the
indicator can preclude accurate Ca2+ measurements. More recently,
click labeling methods for sugars,27 lipids,28 glycans,29 nucleic
acids30 and other metabolites bearing an alkyne moiety have been
reported. Thus, our strategy can be applied to various cellular
components bearing alkyne or cyclooctyne moieties. In such cases,
the chemical Ca2+ indicator will be located at the position of interest.
This work was supported by JST-A*STAR joint international
funding program ‘‘Bioelectronics’’ (S.A.) and ‘‘High-Tech Research
Center’’ project of Waseda University (T.I. and S.T.).
Notes and references
1 D. E. Clapham, Cell, 2007, 131, 1047–1058.
2 M. J. Higley and B. L. Sabatini, Neuron, 2008, 59, 902–913.
3 C. Grienberger and A. Konnerth, Neuron, 2012, 73, 862–885.
4 S. M. Baylor and S. Hollingworth, Prog. Biophys. Mol. Biol., 2011, 105,
162–179.
5 S. Wray, T. Burdyga and K. Noble, Cell Calcium, 2005, 38, 397–407.
6 C. Leclerc, I. Neant, S. E. Webb, A. L. Miller and M. Moreau, Biochim.
Biophys. Acta, Mol. Cell Res., 2006, 1763, 1184–1191.
7 T. Ozawa, H. Yoshimura and S. B. Kim, Anal. Chem., 2013, 85, 590–609.
8 A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams,
M. Ikura and R. Y. Tsien, Nature, 1997, 388, 882–887.
9 J. Nakai, M. Ohkura and K. Imoto, Nat. Biotechnol., 2001, 19, 137–141.
10 T. Terai and T. Nagano, Pfluegers Arch., 2013, 465, 347–359.
11 G. Grynkiewicz, M. Poenie and R. Y. Tsien, J. Biol. Chem., 1985, 260,
3440–3450.
12 A. Minta, J. P. Y. Kao and R. Y. Tsien, J. Biol. Chem., 1989, 264,
8171–8178.
13 M. S. Goligorsky, K. A. Hruska, D. J. Loftus and E. L. Elson, J. Cell.
Physiol., 1986, 128, 466–474.
14 F. Di Virgilio, T. H. Steinberg, J. A. Swanson and S. C. Silverstein,
J. Immunol., 1988, 140, 915–920.
15 F. Di Virgilio, T. H. Steinberg and S. C. Silverstein, Cell Calcium,
1990, 11, 57–62.
Fig. 4 Evaluation of intracellular retention time of the Ca2+ indicators. The
fluorescence images of N3-fura-2 (a) and fura-2 (b) were taken after 6 hours.
(c) Time course of the changes of fluorescence intensity emitted from fura-2, fura-PE3,
and N3-fura-2 loaded HeLa cells was monitored. (d) Baseline drift of the fluorescence
ratio (F340/F380) of fura-2 and N3-fura-2 during a period of 5 min. (e–j) Long-term
changes in Ca2+ transients by histamine detected as the fluorescence ratio recorded at
5 min, 3 hours and 6 hours after loading of N3-fura-2 (e–g) and fura-2 (h–j),
respectively. The addition of histamine is marked by an arrow at t = 0.
16 C. Vorndran, A. Minta and M. Poenie, Biophys. J., 1995, 69,
2112–2124.
17 M. Konishi and M. Watanabe, J. Gen. Physiol., 1995, 106, 1123–1150.
18 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
of fura-2 this phenomenon could not be observed 3 hours and 19 R. Y. Tsien, Nature, 1981, 290, 527–528.
20 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48,
6 hours after loading because the signal was lost in the background
noise (Fig. 4h–j). By contrast, fluorescence ratio changes of N3-fura-2
including cytosolic Ca2+ oscillation could even be observed 6 hours
after loading with a high signal-to-noise ratio (Fig. 4e–g). These
results clearly indicated that N3-fura-2 was retained within the
cytosol and acted as an effective indicator for at least 6 hours.
In conclusion, we have synthesized a novel clickable Ca2+
indicator and achieved ‘‘intracellular copper-free click reaction’’ with
DBCO-modified intracellular proteins. Our Ca2+ indicator displays
an extended cytosolic retention by comparison to fura-2 and
commercially available long-term Ca2+ tracer fura-PE3. Although
the retention time is extended to 6 hours, it is shorter than the
successive expression of fluorescent proteins of several days.25 The
advantage of our method is unnecessity of any troublesome gene
transfection steps. Just the addition of the DBCO reagent and the
indicator to the cells is enough to measure cytosolic Ca2+ oscillation.
Development of this method for long-term Ca2+ tracing will
be invaluable in studying transient changes in the intracellular
6974–6998.
21 S. S. van Berkel, S. Brauch, L. Gabriel, M. Henze, S. Stark, D. Vasilev,
L. A. Wessjohann, M. Abbas and B. Westermann, Angew. Chem., Int.
Ed., 2012, 51, 5343–5346.
22 J. E. B. Gudmundur and G. Haraldsson, Tetrahedron, 1997, 53,
215–224.
23 M. Collot, C. Loukou, A. V. Yakovlev, C. D. Wilms, D. Li, A. Evrard,
A. Zamaleeva, L. Bourdieu, J. F. Leger, N. Ropert, J. Eilers, M. Oheim,
A. Feltz and J. M. Mallet, J. Am. Chem. Soc., 2012, 134, 14923–14931.
24 M. F. Debets, S. S. van Berkel, S. Schoffelen, F. P. Rutjes, J. C. van
Hest and F. L. van Delft, Chem. Commun., 2010, 46, 97–99.
25 M. Ikeda, T. Sugiyama, C. S. Wallace, H. S. Gompf, T. Yoshioka,
A. Miyawaki and C. N. Allen, Neuron, 2003, 38, 253–263.
26 A. Muto, S. Kume, T. Inoue, H. Okano and K. Mikoshiba, J. Cell Biol.,
1996, 135, 181–190.
27 P. Shieh, M. J. Hangauer and C. R. Bertozzi, J. Am. Chem. Soc., 2012,
134, 17428–17431.
28 C. Y. Jao, M. Roth, R. Welti and A. Salic, Proc. Natl. Acad. Sci. U. S. A.,
2009, 106, 15332–15337.
29 A. Salic and T. J. Mitchison, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
2415–2420.
30 M. S. Siegrist, S. Whiteside, J. C. Jewett, A. Aditham, F. Cava and
C. R. Bertozzi, ACS Chem. Biol., 2013, 8, 500–505.
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