Single-Cell Activation of the cAMP-Signaling Pathway in 3D Tissues with FRET-Assisted Two-Photon Activation of bPAC.
Tomoaki KinjoTetsuya WatabeKenju KobachiKenta TeraiMichiyuki MatsudaPublished in: ACS chemical biology (2020)
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 Fürster 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.
Keyphrases
- energy transfer
- single cell
- quantum dots
- living cells
- induced apoptosis
- signaling pathway
- fluorescent probe
- single molecule
- high throughput
- cell cycle arrest
- gene expression
- binding protein
- sensitive detection
- pi k akt
- endoplasmic reticulum stress
- epithelial mesenchymal transition
- stem cells
- small molecule
- cell death
- bone marrow
- photodynamic therapy
- fluorescence imaging