Visual quantification of prostaglandin E 2 discharge from a single cell.

Calcium transients drive cells to discharge prostaglandin E 2 (PGE 2 ). We visualized PGE 2 -induced protein kinase A (PKA) activation and quantitated PGE 2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE 2 -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 PGE 2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer. Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE 2 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 PGE 2 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 PGE 2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE 2 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 PGE 2 .Keywords: prostaglandin E 2 , imaging, intercellular communication, biosensor, quantification.