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FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule

Nature Structural Biology volume 8, pages 1069–1073 (2001)Cite this article

Abstract

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 μM.

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Figure 1: Design of YC6.1.
Figure 2: 1H-15N HSQC NMR spectra of 15N labeled CaM in complex with CKKp and 15N labeled (N-CaM)-CKKp-(C-CaM).
Figure 3: Fluorescence properties of YC6.1 in vitro.
Figure 4: Ca2+ imaging of HeLa cells using YC2.1 and YC6.1.
Figure 5: Ca2+ imaging of rat hippocampus neurons using YC2.1 and YC6.1.

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Acknowledgements

This work was supported in part by a grant from Howard Hughes Medical Institute (HHMI) to M.I., CREST (Core Research for Evolutional Science and Technology) and an NCIC Fellowship to T.K.M.. M.I. is an HHMI International Scholar and a Canadian Institutes of Health Research Scientist. We thank J. Gooding for her excellent assistance in manuscript editing. The YC6 vectors are available from the authors upon request.

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Authors and Affiliations

  1. Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9, Ontario, Canada

    Kevin Truong, Kit I. Tong, Tapas Kumar Mal & Mitsuhiko Ikura

  2. Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, The Institute of Physical and Chemical Science (RIKEN), 2-1 Hirosawa, Wako-city, 351-0198, Saitama, Japan

    Asako Sawano, Hideaki Mizuno, Hiroshi Hama & Atsushi Miyawaki

  3. Brain Science Research Division, Brain Science and Life Technology Research Foundation, 1-28-12 Narimasu, Itabashi, 175-0094, Tokyo, Japan

    Asako Sawano

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  1. Kevin Truong
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Correspondence to Mitsuhiko Ikura.

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Truong, K., Sawano, A., Mizuno, H. et al. FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nat Struct Mol Biol 8, 1069–1073 (2001). https://doi.org/10.1038/nsb728

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