{"title":"单个细胞前列腺素E2放电的视觉定量。","authors":"Tetsuya Watabe, Shinya Yamahira, Michiyuki Matsuda, Kenta Terai","doi":"10.1247/csf.23047","DOIUrl":null,"url":null,"abstract":"<p><p>Calcium transients drive cells to discharge prostaglandin E<sub>2</sub> (PGE<sub>2</sub>). We visualized PGE<sub>2</sub>-induced protein kinase A (PKA) activation and quantitated PGE<sub>2</sub> secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE<sub>2</sub>-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<sub>2</sub> 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 PGE<sub>2</sub> 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<sub>2</sub> 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<sub>2</sub> upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE<sub>2</sub> 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<sub>2</sub>.Key words: prostaglandin E<sub>2</sub>, imaging, intercellular communication, biosensor, quantification.</p>","PeriodicalId":9927,"journal":{"name":"Cell structure and function","volume":" ","pages":"241-249"},"PeriodicalIF":2.0000,"publicationDate":"2023-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11496778/pdf/","citationCount":"0","resultStr":"{\"title\":\"Visual quantification of prostaglandin E<sub>2</sub> discharge from a single cell.\",\"authors\":\"Tetsuya Watabe, Shinya Yamahira, Michiyuki Matsuda, Kenta Terai\",\"doi\":\"10.1247/csf.23047\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Calcium transients drive cells to discharge prostaglandin E<sub>2</sub> (PGE<sub>2</sub>). We visualized PGE<sub>2</sub>-induced protein kinase A (PKA) activation and quantitated PGE<sub>2</sub> secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE<sub>2</sub>-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<sub>2</sub> 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 PGE<sub>2</sub> 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<sub>2</sub> 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<sub>2</sub> upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE<sub>2</sub> 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<sub>2</sub>.Key words: prostaglandin E<sub>2</sub>, imaging, intercellular communication, biosensor, quantification.</p>\",\"PeriodicalId\":9927,\"journal\":{\"name\":\"Cell structure and function\",\"volume\":\" \",\"pages\":\"241-249\"},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2023-12-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11496778/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cell structure and function\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1247/csf.23047\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/10/7 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell structure and function","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1247/csf.23047","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/10/7 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Visual quantification of prostaglandin E2 discharge from a single cell.
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.
期刊介绍:
Cell Structure and Function is a fully peer-reviewed, fully Open Access journal. As the official English-language journal of the Japan Society for Cell Biology, it is published continuously online and biannually in print.
Cell Structure and Function publishes important, original contributions in all areas of molecular and cell biology. The journal welcomes the submission of manuscripts on research areas such as the cell nucleus, chromosomes, and gene expression; the cytoskeleton and cell motility; cell adhesion and the extracellular matrix; cell growth, differentiation and death; signal transduction; the protein life cycle; membrane traffic; and organelles.