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An ultramarine fluorescent protein with increased photostability and pH insensitivity

Abstract

We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concentration and caspase-3 activation in the same apoptotic cells.

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Figure 1: Characterization of UMFP variants.
Figure 2: pH-sensitivity of Sirius.
Figure 3: Dual functional imaging with two FRET pairs.

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Acknowledgements

We thank R.E. Campbell (University of Alberta) and A. Miyawaki (RIKEN) for providing the cDNA encoding EBFP2 and SapRC2, respectively. This work was partly supported by grants from Scientific Research on Advanced Medical Technology of the Ministry of Labor, Health and Welfare of Japan and the Japanese Ministry of Education, Science and Technology.

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Contributions

W.T. performed experiments and data analysis; T.M. and I.K. contributed to gene construction; T.T., To.N. and K.S. set up microscopy systems; K.H. contributed to imaging of phagocytosis; Ta.N. contributed to the conceptual development, experimental design, data analysis and manuscript preparation.

Corresponding author

Correspondence to Takeharu Nagai.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Table 1, Supplementary Notes 1–2 and Supplementary Methods (PDF 2041 kb)

Supplementary Video 1

Imaging the phagocytosis of bacteria by Dictyostelium discoidium. Tracking of bacteria expressing EGFP (green color in left panel) and Sirius (cyan color in right panel) phagocytosed by a Dictyostelium cell. (MOV 3573 kb)

Supplementary Video 2

Dual functional imaging using two FRET-based indicators. Dual FRET ratio imaging of HeLa cells co-expressing two FRET indicators, SC-SCAT3 (left) and SapRC2 (right). (MOV 2915 kb)

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Tomosugi, W., Matsuda, T., Tani, T. et al. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods 6, 351–353 (2009). https://doi.org/10.1038/nmeth.1317

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