SSBD - SSBD:database - Fig3AB_PKA_EGF

Detail of Fig3AB_PKA_EGF_3

SSBD:Repository

Title

Time-lapse FRET images of reporter HeLa cells and producer HeLa cells treated with EGF

Description

Time-lapse FRET images of Booster-PKA in reporter COX-DKO (COX-1 and COX-2 deficient)-HeLa cells cocultured with producer HeLa cells. In the producer cells, optogenetic calcium channel stimulator: OptoSTIM1 (CRY2clust) is expressed, and PGE2 (prostaglandin E2) is generated after blue light flash. It is released and binds to the EP2 on the reporter cells and induces the FRET of PKA biosensor, Booster-PKA. These serial processes reveal the coverage of PGE2 around one activated cell. This dataset includs 5 files of serial imaging. The images of iRFP were obtained at the start and the end of the imaging showing the producer cells. 50 ng/ml EGF was applied 1 hour before imaging.

Release, Updated

2026-01-21

Organism

Homo sapiens ( NCBI:txid9606 )

Strain(s)

Cell Line

HeLa cell

Molecular Function (MF)

Biological Process (BP)

cell communication , signal transduction

Cellular Component (CC)

cytoplasm

Biological Imaging Method

fluorescence microscopy ( Fbbi:00000246 )
time lapse microscopy ( Fbbi:00000249 )
FRET ( Fbbi:00000367 )

X scale

2.7 micrometer

Y scale

2.7 micrometer

Image Acquisition

Experiment type

Microscope type

Acquisition mode

Contrast method

Microscope model

Detector model

Objective model

Filter set

Summary of Methods

Watabe T, Yamahira S, Matsuda M, Terai K. Visual quantification of prostaglandin E2 discharge from a single cell. Cell Struct Funct . 2023 Dec 7;48(2):241-249.

Related paper(s)

Tetsuya Watabe, Shinya Yamahira, Michiyuki Matsuda, Kenta Terai (2023) Visual quantification of prostaglandin E(2) discharge from a single cell., Cell structure and function

Published in 2023 Oct 7 (Electronic publication in Oct. 7, 2023, midnight )

doi: 10.1247/csf.23047
pmid: 37813623

(Abstract) 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 Forster 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.

Contact

Kenta Terai , Kyoto University , Graduate School of Biostudies , Laboratory of Bioimaging and Cell Signaling

Contributors

Tetsuya Watabe, Shinya Yamahira