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Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis
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SYSNO ASEP 0507540 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis Tvůrce(i) Janáček, Jiří (FGU-C) RID, ORCID
Brejchová, Jana (FGU-C) RID, ORCID
Svoboda, Petr (FGU-C) RID, ORCIDZdroj.dok. Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier - ISSN 0005-2736
Roč. 1861, č. 7 (2019), s. 1364-1354Poč.str. 9 s. Jazyk dok. eng - angličtina Země vyd. NL - Nizozemsko Klíč. slova FRAP ; adaptive ROI approach ; delta-Opioid receptor-eYFP mobility ; plasma membrane ; cholesterol depletion ; cholesterol replenishment Vědní obor RIV CE - Biochemie Obor OECD Biochemical research methods CEP GA17-05903S GA ČR - Grantová agentura ČR LM2015062 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy EF16_013/0001775 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy Výzkumná infrastruktura Czech-BioImaging - 90062 - Ústav molekulární genetiky AV ČR, v. v. i. Způsob publikování Open access s časovým embargem (01.06.2020) Institucionální podpora FGU-C - RVO:67985823 UT WOS 000474325100008 EID SCOPUS 85065776933 DOI 10.1016/j.bbamem.2019.04.012 Anotace Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform, however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.
With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (M-f) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.
When this method was used to determine the mobility of the delta-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of delta-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and M-f values of other PM proteins.Pracoviště Fyziologický ústav Kontakt Lucie Trajhanová, lucie.trajhanova@fgu.cas.cz, Tel.: 241 062 400 Rok sběru 2020 Elektronická adresa https://doi.org/10.1016/j.bbamem.2019.04.012
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