ChemComm
Communication
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.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 7313--7315 7315