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Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin


Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators ‘cameleons’. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP)1,2, calmodulin3,4,5, the calmodulin-binding peptide M13 (ref. 6), and an enhanced green- or yellow-emitting GFP7,8,9. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs2. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10−8 to 10−2 M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 µM at rest, and 1 to 50 µM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

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Figure 1: Schematic structures and sequences of cameleons.
Figure 2: Properties of cameleons in vitro.
Figure 3: Imaging of cameleons in HeLa cells.
Figure 4: GFP-based detection of protein association/dissociation.

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  1. Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and post-translational autooxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, 12501–12504 (1994).

    Google Scholar 

  2. Heim, R. & Tsien, R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence energy transfer. Curr. Biol. 6, 178–182 (1996).

    Google Scholar 

  3. Crivici, A. & Ikura, M. Molecular and structural basis of target recognition by calmodulin. Annu. Rev. Biophys. Biomol. Struct. 24, 85–116 (1995).

    Google Scholar 

  4. Babu, Y. S., Bugg, C. E. & Cook, W. J. Structure of calmodulin refined at 2.2 Å resolution. J. Mol. Biol. 204, 191–204 (1988).

    Google Scholar 

  5. Falke, J. J., Drake, S. K., Hazard, A. L. & Peersen, O. B. Molecular tuning of ion binding to calcium signaling proteins. Q. Rev. Biophys. 27, 219–290 (1994).

    Google Scholar 

  6. Ikura, al. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992).

    Google Scholar 

  7. Heim, R., Cubitt, A. B. & Tsien, R. Y. Improved green fluorescence. Nature 373, 663–664 (1995).

    Article  ADS  CAS  Google Scholar 

  8. Cormack, B. P., Valdivia, R. H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Google Scholar 

  9. Ormö, al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Google Scholar 

  10. Grynkiewicz, G., Poenie, . & Tsien, R. Y. Anew generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    Google Scholar 

  11. Tse, F. W., Tse, A. & Hille, B. Cyclic Ca2+ changes in intracellular stores of gonadotropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations. Proc. Natl Acad. Sci. USA 91, 9750–9754 (1994).

    Google Scholar 

  12. Hofer, A. M. & Schulz, I. Quantification of intraluminal free [Ca] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: some considerations. Cell Calcium 20, 235–242 (1996).

    Google Scholar 

  13. Golovina, V. A. & Blaustein, M. P. Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum. Science 275, 1643–1648 (1997).

    Google Scholar 

  14. Montero, al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 14, 5467–5475 (1995).

    Google Scholar 

  15. Kendall, J. M., Badminton, M. N., Sala-Newby, G. B., Campbell, A. K. & Rembold, C. M. Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells. Biochem. J. 318, 383–387 (1996).

    Google Scholar 

  16. Porumb, T., Yau, P., Harvey, T. S. & Ikura, M. Acalmodulin-target peptide hybrid molecule with unique calcium-binding properties. Protein Eng. 7, 109–115 (1994).

    Google Scholar 

  17. Maune, J. F., Klee, C. B. & Beckingham, K. Ca2+ binding and conformational change in two series of point mutations to the individual Ca2+-binding sites of calmodulin. J. Biol. Chem. 267, 5286–5296 (1992).

    Google Scholar 

  18. Gao, Z. al. Activation of four enzymes by two series of calmodulin mutants with point mutations in individual Ca2+ binding sites. J. Biol. Chem. 268, 20096–20104 (1993).

    Google Scholar 

  19. Martin, S. al. Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule. Biochemistry 35, 3508–3517 (1996).

    Google Scholar 

  20. Zolotukhin, S., Potter, M., Hauswirth, W., Guy, J. & Muzyczka, N. A“humanized” green fluorescent protein cDNA adapted for high levels of expression in mammalian cells. J. Virol. 70, 4646–4654 (1996).

    Google Scholar 

  21. Smit, M. al. Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5′-nucleotide receptor. Eur. J. Pharmacol. 247, 223–226 (1993).

    Google Scholar 

  22. Bootman, M. D., Cheek, T. R., Moreton, R. B., Bennett, D. L. & Berridge, M. J. Smoothly graded Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. J. Biol. Chem. 269, 24783–24791 (1994).

    Google Scholar 

  23. Brini, M., Marsault, R., Bastianutto, C., Pozzan, T. & Rizzuto, R. Nuclear targeting of aequorin. A new approach for measuring nuclear Ca2+ concentration in intact cells. Cell Calcium 16, 259–268 (1994).

    Google Scholar 

  24. Kendall, J. M., Dormer, R. L. & Campbell, A. K. Targeting aequorin to the endoplasmic reticulum of living cells. Biochem. Biophys. Res. Commun. 189, 1008–1016 (1992).

    Google Scholar 

  25. Bygrave, F. L. & Benedetti, A. What is the concentration of calcium ions in the endoplasmic reticulum? Cell Calcium 19, 547–551 (1996).

    Google Scholar 

  26. Button, D. & Eidsath, A. Aequorin targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or InsP3-induced release of Ca++. Mol. Biol. Cell 7, 419–434 (1996).

    Google Scholar 

  27. Romoser, V. A., Hinkle, P. M. & Persechini, A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. J. Biol. Chem. 272, 13270–13274 (1997).

    Google Scholar 

  28. Kozak, M. The scanning model for translation: an update. J. Cell Biol. 108, 229–241 (1989).

    Google Scholar 

  29. Adams, S. R., Bacskai, B. J., Taylor, S. S. & Tsien, R. Y. Fluorescent Probes for Biological Activity of Living Cells–A Practical Guide(ed. Mason, W. T.) 133–149 (Academic, New York, (1993)).

    Google Scholar 

  30. Balch, W. E., McCaffery, J. M., Plutner, H. & Farquhar, M. G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 75, 841–852 (1994).

    Google Scholar 

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We thank S. R. Adams and A. B. Cubitt for advice, C. Zuker for the GFP antibody, and C. Klee for the original Xenopus calmodulin gene. This work was supported by HHMI (R.Y.T.), NIH (R.Y.T.), HFSP (R.Y.T. and A.M.), MRCC (M.I.) and the Spanish Ministry of Science (J.L.). M.I. is an HHMI international research scholar and MRCC scholar.

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Miyawaki, A., Llopis, J., Heim, R. et al. Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

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