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

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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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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