Detail of FigureS2_HeLa_4526NES_FSK



Project
Title
Fluorescence lifetime images of HeLa cells expressing FRET biosensors before and after addition of forskolin.
Description
Fluorescence lifetime images of HeLa cells expressing FRET biosensors before and after addition of forskolin.
Release, Updated
2022-03-31
License
CC BY
Kind
Image data
File Formats
.tif
Data size
468.7 KB

Organism
Homo sapiens ( NCBITaxon:9606 )
Strain(s)
-
Cell Line
HeLa cell ( CLO_0003684 )

Datatype
-
Molecular Function (MF)
extracellular signal-regulated kinase activity ( GO:0004707 )
Biological Process (BP)
-
Cellular Component (CC)
-
Biological Imaging Method
differences in fluorescence lifetime ( Fbbi:00000605 )
X scale
-
Y scale
-
Z scale
-
T scale
-

Image Acquisition
Experiment type
-
Microscope type
-
Acquisition mode
-
Contrast method
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Microscope model
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Detector model
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Objective model
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Filter set
-

Summary of Methods
See details in Watabe T, et. al. (2020) ACS Sens., 5(3):719-730.
Related paper(s)

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 )

(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.
(MeSH Terms)

Contact
Kenta Terai , Graduate School of Medicine, Kyoto University , Department of Pathology and Biology of Diseases
Contributors

OMERO Dataset
OMERO Project
Source