Visual quantification of prostaglandin E2 discharge from a single cell.

Abstract

Calcium transients drive cells to discharge prostaglandin E₂ (PGE₂). We visualized PGE₂-induced protein kinase A (PKA) activation and quantitated PGE₂ secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE₂-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE₂ reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE₂ to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE₂ diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE₂ upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE₂ discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE₂.Key words: prostaglandin E₂, imaging, intercellular communication, biosensor, quantification.