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Detail of Figure5A_FSK_HeLa_FSKtiteration1_w5DIC-fast

Tetsuya Watabe, Kenta Terai, Kenta Sumiyama, Michiyuki Matsuda (2020) Booster, a Red-Shifted Genetically Encoded Forster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools., ACS sensors, Volume 5, Number 3, pp. 719-730

Published in 2020 Mar 27 (Electronic publication in Feb. 26, 2020, midnight )

• doi: 10.1021/acssensors.9b01941
• pmid: 32101394
  

(Abstract) Genetically encoded Forster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOkappa and mKate2 as the favorable donor and acceptor pair by calculating the Forster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.

• Adenylyl Cyclases
• Animals
• Bacterial Proteins
• Biosensing Techniques[major]
• Cyclic AMP-Dependent Protein Kinases/genetics
• Cyclic AMP-Dependent Protein Kinases/metabolism[major]
• Dogs
• Female
• Fluorescence Resonance Energy Transfer[major]
• Green Fluorescent Proteins
• HEK293 Cells
• HeLa Cells
• Humans
• Light
• Luminescent Proteins
• Madin Darby Canine Kidney Cells
• Male
• Mice, Transgenic
• Optogenetics
• Spectrometry, Fluorescence