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Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

SYSNO ASEP	0507540
Document Type	J - Journal Article
R&D Document Type	Journal Article
Subsidiary J	Článek ve WOS
Title	Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis
Author(s)	Janáček, Jiří (FGU-C)_{RID, ORCID} Brejchová, Jana (FGU-C)_{RID, ORCID} Svoboda, Petr (FGU-C)_{RID, ORCID}
Source Title	Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier - ISSN 0005-2736 Roč. 1861, č. 7 (2019), s. 1364-1354
Number of pages	9 s.
Language	eng - English
Country	NL - Netherlands
Keywords	FRAP ; adaptive ROI approach ; delta-Opioid receptor-eYFP mobility ; plasma membrane ; cholesterol depletion ; cholesterol replenishment
Subject RIV	CE - Biochemistry
OECD category	Biochemical research methods
R&D Projects	GA17-05903S GA ČR - Czech Science Foundation (CSF)
	LM2015062 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
	EF16_013/0001775 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
Research Infrastructure	Czech-BioImaging - 90062 - Ústav molekulární genetiky AV ČR, v. v. i.
Method of publishing	Open access with time embargo (01.06.2020)
Institutional support	FGU-C - RVO:67985823
UT WOS	000474325100008
EID SCOPUS	85065776933
DOI	10.1016/j.bbamem.2019.04.012
Annotation	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.
Workplace	Institute of Physiology
Contact	Lucie Trajhanová, lucie.trajhanova@fgu.cas.cz, Tel.: 241 062 400
Year of Publishing	2020
Electronic address	https://doi.org/10.1016/j.bbamem.2019.04.012

Number of the records: 1