3.67 second for each time interval, 3.74 second for each time interval, 4.08 second for each time interval, 3.48 second for each time interval, 2.62 second for each time interval, 2.63 second for each time interval, 2.60 second for each time interval, 4 second for each time interval, 3.76 second for each time interval, 3.64 second for each time interval, 3.59 second for each time interval, 2.59 second for each time interval, 3.61 second for each time interval, 3.55 second for each time interval, 2.68 second for each time interval, 120 second for each time interval, 2.55 second for each time interval
Image Acquisition
Experiment type
TimeLapse
Microscope type
ConfocalMicroscope
Acquisition mode
LaserScanningConfocalMicroscopy
Contrast method
Fluorescence
Microscope model
Olympus IX81 FV1000
Detector model
-
Objective model
Olympus UPLANSAPO 60xO/N.A. 1.35
Filter set
-
Related paper(s)
Youichi Uda, Yuhei Goto, Shigekazu Oda, Takayuki Kohchi, Michiyuki Matsuda, Kazuhiro Aoki (2017) Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling., Proceedings of the National Academy of Sciences of the United States of America, Volume 114, Number 45, pp. 11962-11967
Published in 2017 Nov 7
(Electronic publication in Oct. 24, 2017, midnight )
(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.