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Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):11962-11967. doi: 10.1073/pnas.1707190114. Epub 2017 Oct 24.

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.

Uda Y1,2,3, Goto Y2,3, Oda S2,3, Kohchi T4, Matsuda M1,5, Aoki K6,3,7,8.

Author information

1
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
2
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
3
Division of Quantitative Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
4
Laboratory of Plant Molecular Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
5
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
6
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; k-aoki@nibb.ac.jp.
7
Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
8
Department of Basic Biology, Faculty of Life Science, SOKENDAI (Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.

Abstract

Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.

KEYWORDS:

ERK; FRET; cell signaling; optogenetics; phytochrome

PMID:
29078307
PMCID:
PMC5692546
DOI:
10.1073/pnas.1707190114
[Indexed for MEDLINE]
Free PMC Article

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