Expanded Palette of Nano-lanterns for Real-time Multicolor Luminescence Imaging

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2015 Apr 2

PMID 25831507

Citations 64

Authors

Akira Takai

Masahiro Nakano

Kenta Saito

Remi Haruno

Tomonobu M Watanabe

Tatsuya Ohyanagi

Takashi Jin

Yasushi Okada

Takeharu Nagai

Affiliations

Soon will be listed here.

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.

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References

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H . Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008; 132(3):487-98. DOI: 10.1016/j.cell.2007.12.033. View

Saito K, Chang Y, Horikawa K, Hatsugai N, Higuchi Y, Hashida M . Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun. 2012; 3:1262. PMC: 3535334. DOI: 10.1038/ncomms2248. View

Takai A, Inomata H, Arakawa A, Yakura R, Matsuo-Takasaki M, Sasai Y . Anterior neural development requires Del1, a matrix-associated protein that attenuates canonical Wnt signaling via the Ror2 pathway. Development. 2010; 137(19):3293-302. DOI: 10.1242/dev.051136. View

Zhao H, Doyle T, Wong R, Cao Y, Stevenson D, Piwnica-Worms D . Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals. Mol Imaging. 2004; 3(1):43-54. DOI: 10.1162/15353500200403181. View

Akerboom J, Carreras Calderon N, Tian L, Wabnig S, Prigge M, Tolo J . Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci. 2013; 6:2. PMC: 3586699. DOI: 10.3389/fnmol.2013.00002. View

Metzger M, Maurer M, Dancy B, Michaelis S . Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery. J Biol Chem. 2008; 283(47):32302-16. PMC: 2583311. DOI: 10.1074/jbc.M806424200. View

Miyawaki A . Bringing bioluminescence into the picture. Nat Methods. 2007; 4(8):616-7. DOI: 10.1038/nmeth0807-616. View

Loening A, Wu A, Gambhir S . Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat Methods. 2007; 4(8):641-3. DOI: 10.1038/nmeth1070. View

Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink M . Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun. 2012; 3:751. PMC: 3316892. DOI: 10.1038/ncomms1738. View

10.

Loening A, Fenn T, Wu A, Gambhir S . Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel. 2006; 19(9):391-400. DOI: 10.1093/protein/gzl023. View

11.

Fukuda N, Matsuda T, Nagai T . Optical control of the Ca2+ concentration in a live specimen with a genetically encoded Ca2+-releasing molecular tool. ACS Chem Biol. 2014; 9(5):1197-203. DOI: 10.1021/cb400849n. View

12.

Chu J, Haynes R, Corbel S, Li P, Gonzalez-Gonzalez E, Burg J . Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat Methods. 2014; 11(5):572-8. PMC: 4008650. DOI: 10.1038/nmeth.2888. View

13.

Fuerer C, Nusse R . Lentiviral vectors to probe and manipulate the Wnt signaling pathway. PLoS One. 2010; 5(2):e9370. PMC: 2826402. DOI: 10.1371/journal.pone.0009370. View

14.

Nagai T, Sawano A, Park E, Miyawaki A . Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A. 2001; 98(6):3197-202. PMC: 30630. DOI: 10.1073/pnas.051636098. View

15.

Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M . A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell. 2004; 7(1):133-44. DOI: 10.1016/j.devcel.2004.06.005. View

16.

Shcherbakova D, Hink M, Joosen L, Gadella T, Verkhusha V . An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J Am Chem Soc. 2012; 134(18):7913-23. PMC: 3348967. DOI: 10.1021/ja3018972. View

17.

Nagai T, Ibata K, Park E, Kubota M, Mikoshiba K, Miyawaki A . A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol. 2001; 20(1):87-90. DOI: 10.1038/nbt0102-87. View

18.

Magidson V, Khodjakov A . Circumventing photodamage in live-cell microscopy. Methods Cell Biol. 2013; 114:545-60. PMC: 3843244. DOI: 10.1016/B978-0-12-407761-4.00023-3. View

19.

Balaskas N, Ribeiro A, Panovska J, Dessaud E, Sasai N, Page K . Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube. Cell. 2012; 148(1-2):273-84. PMC: 3267043. DOI: 10.1016/j.cell.2011.10.047. View

20.

Miranda J, Weaver A, Qin Y, Park J, Stoddard C, Lin M . New alternately colored FRET sensors for simultaneous monitoring of Zn²⁺ in multiple cellular locations. PLoS One. 2012; 7(11):e49371. PMC: 3500285. DOI: 10.1371/journal.pone.0049371. View