Detail of Figure1B_mTFP1_HeLa

Fluorescence lifetime images of Hela cells expressing mTFP1 alone.
Fluorescence lifetime images of Hela cells expressing mTFP1 alone.
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Homo sapiens ( NCBITaxon:9606 )
Cell Line
HeLa cell ( CLO_0003684 )
Protein names

Molecular Function (MF)
Biological Process (BP)
cAMP-mediated signaling ( GO:0019933 )
Cellular Component (CC)
cAMP-dependent protein kinase complex ( GO:0005952 )
Biological Imaging Method
differences in fluorescence lifetime ( Fbbi:00000605 )
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T scale

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

Summary of Methods
See details in Kinjo T, et. al. (2020) ACS Chem Biol., 15(11):2848-2853.
Related paper(s)

Tomoaki Kinjo, Tetsuya Watabe, Kenju Kobachi, Kenta Terai, Michiyuki Matsuda (2020) Single-Cell Activation of the cAMP-Signaling Pathway in 3D Tissues with FRET-Assisted Two-Photon Activation of bPAC., ACS chemical biology, Volume 15, Number 11, pp. 2848-2853

Published in 2020 Nov 20 (Electronic publication in Oct. 19, 2020, midnight )

(Abstract) Bacterial photoactivated adenylyl cyclase (bPAC) has been widely used in signal transduction research. However, due to its low two-photon absorption, bPAC cannot be efficiently activated by two-photon (2P) excitation. Taking advantage of the high two-photon absorption of monomeric teal fluorescent protein 1 (mTFP1), we herein developed 2P-activatable bPAC (2pabPAC), a fusion protein consisting of bPAC and mTFP1. In 2pabPAC, the energy absorbed by mTFP1 excites bPAC by Furster resonance energy transfer (FRET) at ca. 43% efficiency. The light-induced increase in cAMP was monitored by a red-shifted FRET biosensor for PKA. In 3D MDCK cells and mouse liver, PKA was activated at single-cell resolution under a 2P microscope. We found that PKA activation in a single hepatocyte caused PKA activation in neighboring cells, indicating the propagation of PKA activation. Thus, 2pabPAC will provide a versatile platform for controlling the cAMP signaling pathway and investigating cell-to-cell communication in vivo.
(MeSH Terms)

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

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