Single-Cell Activation of the cAMP-Signaling Pathway in 3D Tissues with FRET-Assisted Two-Photon Activation of bPAC

ACS Chem Biol. 2020 Nov 20;15(11):2848-2853. doi: 10.1021/acschembio.0c00333. Epub 2020 Oct 19.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Green Fluorescent Proteins
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Adenylyl Cyclases