Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F67985823%3A_____%2F19%3A00507540" target="_blank" >RIV/67985823:_____/19:00507540 - isvavai.cz</a>
Result on the web
<a href="https://doi.org/10.1016/j.bbamem.2019.04.012" target="_blank" >https://doi.org/10.1016/j.bbamem.2019.04.012</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1016/j.bbamem.2019.04.012" target="_blank" >10.1016/j.bbamem.2019.04.012</a>
Alternative languages
Result language
angličtina
Original language name
Determination of delta-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis
Original language description
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. nWith 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. nWhen 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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10609 - Biochemical research methods
Result continuities
Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)
Others
Publication year
2019
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Biochimica Et Biophysica Acta-Biomembranes
ISSN
0005-2736
e-ISSN
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Volume of the periodical
1861
Issue of the periodical within the volume
7
Country of publishing house
NL - THE KINGDOM OF THE NETHERLANDS
Number of pages
9
Pages from-to
1364-1354
UT code for WoS article
000474325100008
EID of the result in the Scopus database
2-s2.0-85065776933