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

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10609 - Biochemical research methods

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Biochimica Et Biophysica Acta-Biomembranes

ISSN

0005-2736

e-ISSN

—

Svazek periodika

1861

Číslo periodika v rámci svazku

7

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

1364-1354

Kód UT WoS článku

000474325100008

EID výsledku v databázi Scopus

2-s2.0-85065776933