title: Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.
abstract: Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.
authors.affiliation: Laboratories for Cell Polarity Regulation.
authors.forename: Akira
authors.initials: A
authors.lastname: Takai
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakano
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and.
authors.forename: Kenta
authors.initials: K
authors.lastname: Saito
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and.
authors.forename: Remi
authors.initials: R
authors.lastname: Haruno
authors.affiliation: Comprehensive Bioimaging, PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan.
authors.forename: Tomonobu M
authors.initials: TM
authors.lastname: Watanabe
authors.affiliation: Nano-Bio Probes, and.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ohyanagi
authors.affiliation: Nano-Bio Probes, and.
authors.forename: Takashi
authors.initials: T
authors.lastname: Jin
authors.affiliation: Laboratories for Cell Polarity Regulation, ng1@sanken.osaka-u.ac.jp y.okada@riken.jp.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okada
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan Cell Dynamics Observation, Quantitative Biology Center, RIKEN, Osaka 565-0874, Japan; ng1@sanken.osaka-u.ac.jp y.okada@riken.jp.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004285:Dogs; D053595:Embryonic Stem Cells / Q000166:cytology; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D022001:Focal Adhesions; D005786:Gene Expression Regulation; D008156:Luciferases / Q000737:chemistry*; D049410:Luciferases, Renilla / Q000378:metabolism; D049449:Luminescence; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D020675:Peroxisomes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D048790:Renilla; D016596:Vinculin / Q000737:chemistry
chemical_list: D008164:Luminescent Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D004247:DNA; D008156:Luciferases; C581563:nano-lantern protein; D049410:Luciferases, Renilla; D002118:Calcium
keywords: bioluminescence resonance energy transfer; live imaging; luciferase; luminescence imaging; luminescent indicator
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