Five colour variants of bright luminescent protein for real-time multicolour bioimaging.

Kazushi Suzuki,Guirong Bai,Masahiro Nakano,Matthew J Daniels,Yoshiyuki Arai,Hajime Shinoda,Taichi Kimura,Takeharu Nagai

doi:10.1038/ncomms13718

Kazushi Suzuki, Guirong Bai + Show 6 more

Open Access

https://doi.org/10.1038/ncomms13718

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Journal: Nature Communications	Publication Date: Dec 1, 2016
Citations: 193	License type: CC BY 4.0

Affiliation: Osaka University, John Radcliffe Hospital

Abstract

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca2+ indicator with a 500% signal change, which can image spontaneous Ca2+ dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.

Highlights

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied
We speculated that the unstructured residues of Nluc and fluorescent proteins (FPs) could work as linkers and be deleted without disrupting the function of either protein
We made a library of Nluc and FPs with different linker lengths by systematically truncating the N terminus of Nluc and C terminus of FPs, as well as randomizing two residues at the junction, derived from a KpnI site (Supplementary Figs 1–6)

Summary

Introduction

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. We develop an eNL-based Ca2 þ indicator with a 500% signal change, which can image spontaneous Ca2 þ dynamics in cardiomyocyte and neural cell models These eNL probes facilitate multicolour imaging in living cells and sensitive imaging of a wide repertoire of proteins, even at very low expression levels. Colour variants of luciferase are typically developed by rationally altering residues in the active pocket, which alters the chemical form of the excited luminescent substrate, thereby allowing a colour-shifted emission spectrum[7]. This approach has not proven effective for Nluc owing to the modified chemical structure of furimazine

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