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

0507540 - FGÚ 2020 RIV NL eng J - Journal Article
Janáček, Jiří - Brejchová, Jana - Svoboda, Petr
Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis.
Biochimica Et Biophysica Acta-Biomembranes. Roč. 1861, č. 7 (2019), s. 1364-1354. ISSN 0005-2736. E-ISSN 1879-2642
R&D Projects: GA ČR(CZ) GA17-05903S; GA MŠMT(CZ) LM2015062; GA MŠMT(CZ) EF16_013/0001775
Research Infrastructure: Czech-BioImaging - 90062
Institutional support: RVO:67985823
Keywords : FRAP * adaptive ROI approach * delta-Opioid receptor-eYFP mobility * plasma membrane * cholesterol depletion * cholesterol replenishment
OECD category: Biochemical research methods
Impact factor: 3.411, year: 2019
Method of publishing: Open access with time embargo
https://doi.org/10.1016/j.bbamem.2019.04.012

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.
Permanent Link: http://hdl.handle.net/11104/0298518