SSBD:database

Detail of Figure3C_20200715_4493NES_2nd_pre

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 )

• doi: 10.1021/acschembio.0c00333
• pmid: 33074647
  

(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.

• Adenylyl Cyclases/metabolism
• Animals
• Bacteria/enzymology
• Biosensing Techniques/methods
• Cell Communication
• Cyclic AMP/metabolism[major]
• Cyclic AMP-Dependent Protein Kinases/metabolism[major]
• Dogs
• Enzyme Activation
• Fluorescence Resonance Energy Transfer/methods[major]
• Fluorescent Dyes/metabolism
• Green Fluorescent Proteins/metabolism
• HeLa Cells
• Humans
• Liver/metabolism
• Madin Darby Canine Kidney Cells
• Mice
• Signal Transduction[major]
• Single-Cell Analysis/methods[major]