リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

大学・研究所にある論文を検索できる 「Visual quantification of prostaglandin E₂ discharge from a single cell」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

コピーが完了しました

URLをコピーしました

論文の公開元へ論文の公開元へ
書き出し

Visual quantification of prostaglandin E₂ discharge from a single cell

Watabe, Tetsuya Yamahira, Shinya Matsuda, Michiyuki Terai, Kenta 京都大学 DOI:10.1247/csf.23047

2023

概要

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.

この論文で使われている画像

参考文献

Fischer Scientific) supplemented with 10% FBS, 0.1% w/v bovine

Adachi, T., Kakuta, S., Aihara, Y., Kamiya, T., Watanabe, Y., Osakabe,

serum albumin (BSA; Sigma-Aldrich), 1% v/v GlutaMAX (Thermo

N., Hazato, N., Miyawaki, A., Yoshikawa, S., Usami, T., Karasuyama,

Fischer Scientific), and penicillin/streptomycin. At 5 h after the

H., Kimoto-Nira, H., Hirayama, K., and Tsuji, N.M. 2016. Visualization

medium change, the culture media were replaced with

of probiotic-mediated Ca

FluoroBright DMEM supplemented with 0.1% w/v BSA, 1% v/v

GlutaMAX,

penicillin/streptomycin,

and

μM

CNO.

2+

signaling in intestinal epithelial cells in

vivo. Front. Immunol., 7: 601.

After

Camacho, M., Gerboles, E., Escudero, J.R., Anton, R., Garcia-Moll, X.,

incubation for the designated amount of time, the culture media

and Vila, L. 2007. Microsomal prostaglandin E synthase-1, which is

were collected. The time zero sample was obtained from cells

not coupled to a particular cyclooxygenase isoenzyme, is essential

treated without CNO. PGE2 concentrations in culture media were

for prostaglandin E(2) biosynthesis in vascular smooth muscle

quantified by a colorimetric competitive enzyme immunoassay

cells. J Thromb Haemost, 5: 1411–1419.

kit according to the manufacturer’s protocol (ADI-900-001; Enzo

Clapham, D.E. 2007. Calcium signaling. Cell, 131: 1047–1058.

Life Sciences, Farmingdale, NY, USA).

Clark, J.D., Schievella, A.R., Nalefski, E.A., and Lin, L.L. 1995. Cytosolic

Statistical analysis

Denning, G.M., Figard, P.H., and Spector, A.A. 1982. Effect of fatty

phospholipase A2. J. Lipid. Med. Cell Signal., 12: 83–117.

All statistical analyses and visualizations were performed in

acid modification on prostaglandin production by cultured 3T3

Python using the libraries NumPy, pandas, SciPy, pingouin,

cells. J. Lipid Res., 23: 584–596.

matplotlib, and seaborn. No statistical analysis was used to

predetermine the sample size.

Endo, S., Nomura, T., Chan, B.S., Lu, R., Pucci, M.L., Bao, Y., and

Schuster, V.L. 2002. Expression of PGT in MDCK cell monolayers:

polarized apical localization and induction of active PG transport.

Data availability

Am. J. Physiol. Renal Physiol., 282: F618–F622.

The data that support the findings of this study are available

within the article and its Supplementary Information or from the

Engelke, M., Engels, N., Dittmann, K., Stork, B., and Wienands, J.

2007.

Ca(2+)

signaling

in

antigen

receptor-activated

lymphocytes. Immunol. Rev., 218: 235–246.

corresponding author upon reasonable request.

Evans, J.H., Spencer, D.M., Zweifach, A., and Leslie, C.C. 2001.

Intracellular calcium signals regulating cytosolic phospholipase A2

Author Contributions

translocation to internal membranes. J. Biol. Chem., 276: 30150–

Conceptualization, methodology, validation, formal analysis: T.W.,

K.T.; Investigation: T.W., S.Y. and K.T.; Data curation: T.W., S.Y., and

K.T.; Resources: T.W., K.T. and M.M.; Writing – original draft: T.W.;

Shimizu, T. 1999. Critical duration of intracellular Ca2+ response

Writing – review & editing: K.T. and M.M.; Supervision: K.T. and

required for continuous translocation and activation of cytosolic

M.M.; Project administration: K.T. and M.M.; Funding acquisition:

T.W., K.T. and M.M.

248

30160.

Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T., Iino, M., Itoh, H., and

phospholipase A2. J. Biol. Chem., 274: 5163–5169.

Kalinski, P. 2012. Regulation of immune responses by prostaglandin

Cell Structure and Function 48: 241–249 (2023)

https://doi.org/10.1247/csf.23047

Prostaglandin E2 discharge from a single cell

Cell Structure and Function

E2. J. Immunol., 188: 21–28.

Park, J.Y., Pillinger, M.H., and Abramson, S.B. 2006. Prostaglandin E2

Kanai, N., Lu, R., Satriano, J.A., Bao, Y., Wolkoff, A.W., and Schuster,

V.L. 1995. Identification and characterization of a prostaglandin

transporter. Science, 268: 866–869.

synthesis and secretion: the role of PGE2 synthases. Clin.

Immunol., 119: 229–240.

Petrášek, Z. and Schwille, P. 2008. Precise measurement of diffusion

Lee, S., Park, H., Kyung, T., Kim, N.Y., Kim, S., Kim, J., and Heo, W.D.

2014. Reversible protein inactivation by optogenetic trapping in

cells. Nat. Methods, 11: 633–636.

coefficients using scanning fluorescence correlation spectroscopy.

Biophys. J., 94: 1437–1448.

Regan, J.W. 2003. EP2 and EP4 prostanoid receptor signaling. Life

Leslie, C.C. 2015. Cytosolic phospholipase A2: physiological function

and role in disease. J. Lipid Res., 56: 1386–1402.

Sci., 74: 143–153.

Reid, G., Wielinga, P., Zelcer, N., van der Heijden, I., Kuil, A., de Haas,

Liu, Y., Mi, Y., Mueller, T., Kreibich, S., Williams, E.G., Van Drogen, A.,

M., Wijnholds, J., and Borst, P. 2003. The human multidrug

Borel, C., Frank, M., Germain, P.L., Bludau, I., Mehnert, M., Seifert,

resistance protein MRP4 functions as a prostaglandin efflux

M., Emmenlauer, M., Sorg, I., Bezrukov, F., Bena, F.S., Zhou, H.,

transporter and is inhibited by nonsteroidal antiinflammatory drugs.

Dehio, C., Testa, G., Saez-Rodriguez, J., Antonarakis, S.E., Hardt,

Proc. Natl. Acad. Sci. U.S.A., 100: 9244–9249.

W.D., and Aebersold, R. 2019. Multi-omic measurements of

heterogeneity in HeLa cells across laboratories. Nat. Biotechnol.,

37: 314–322.

Schuster, V.L. 2002. Prostaglandin transport. Porstag. Oth. Lipid

Mediat., 68–69: 633–647.

Shiraya, K., Hirata, T., Hatano, R., Nagamori, S., Wiriyasermkul, P.,

Miah, M.F., Conseil, G., and Cole, S.P. 2016. N-linked glycans do not

Jutabha, P., Matsubara, M., Muto, S., Tanaka, H., Asano, S., Anzai, N.,

affect plasma membrane localization of multidrug resistance

Endou, H., Yamada, A., Sakurai, H., and Kanai, Y. 2010. A novel

protein 4 (MRP4) but selectively alter its prostaglandin E2

transporter of SLC22 family specifically transports prostaglandins

transport activity. Biochem. Biophys. Res. Commun., 469: 954–959.

and co-localizes with 15-hydroxyprostaglandin dehydrogenase in

Murata, T., Honda, T., Egawa, G., Yamamoto, Y., Ichijo, R., Toyoshima,

F., Dainichi, T., and Kabashima, K. 2018. Transient elevation of

cytoplasmic calcium ion concentration at a single cell level

precedes morphological changes of epidermal keratinocytes

during cornification. Sci. Rep., 8: 6610.

Smith, W.L. and Langenbach, R. 2001. Why there are two

cyclooxygenase isozymes. J. Clin. Invest., 107: 1491–1495.

Watabe, T., Yamahira, S., Takakura, K., Thumkeo, D., Narumiya, S.,

Matsuda, M., and Terai, K. 2023. Calcium transients trigger switch-

Narumiya, S. 2007. Physiology and pathophysiology of prostanoid

receptors. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci., 83: 296–319.

Cell Structure and Function 48: 241–249 (2023)

renal proximal tubules. J. Biol. Chem., 285: 22141–22151.

like discharge of prostaglandin E2 (PGE2) in an ERK-dependent

manner. eLife, 12: RP86727.

https://doi.org/10.1247/csf.23047

249

...

参考文献をもっと見る

全国の大学の
卒論・修論・学位論文

一発検索!

この論文の関連論文を見る