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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 ASEP0507540
    Druh ASEPJ - Článek v odborném periodiku
    Zařazení RIVJ - Článek v odborném periodiku
    Poddruh JČlánek ve WOS
    NázevDetermination 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, ORCID
    Zdroj.dok.Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier - ISSN 0005-2736
    Roč. 1861, č. 7 (2019), s. 1364-1354
    Poč.str.9 s.
    Jazyk dok.eng - angličtina
    Země vyd.NL - Nizozemsko
    Klíč. slovaFRAP ; adaptive ROI approach ; delta-Opioid receptor-eYFP mobility ; plasma membrane ; cholesterol depletion ; cholesterol replenishment
    Vědní obor RIVCE - Biochemie
    Obor OECDBiochemical research methods
    CEPGA17-05903S GA ČR - Grantová agentura ČR
    LM2015062 GA MŠk - Ministerstvo školství, mládeže a tělovýchovy
    EF16_013/0001775 GA MŠk - Ministerstvo školství, mládeže a tělovýchovy
    Výzkumná infrastrukturaCzech-BioImaging - 90062 - Ústav molekulární genetiky AV ČR, v. v. i.
    Způsob publikováníOpen access s časovým embargem (01.06.2020)
    Institucionální podporaFGU-C - RVO:67985823
    UT WOS000474325100008
    EID SCOPUS85065776933
    DOI10.1016/j.bbamem.2019.04.012
    AnotaceFluorescence 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.
    PracovištěFyziologický ústav
    KontaktLucie Trajhanová, lucie.trajhanova@fgu.cas.cz, Tel.: 241 062 400
    Rok sběru2020
    Elektronická adresahttps://doi.org/10.1016/j.bbamem.2019.04.012
Počet záznamů: 1