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 (Mf) 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 δ-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 δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.
- MeSH
- artefakty MeSH
- buněčná membrána metabolismus MeSH
- cholesterol metabolismus MeSH
- FRAP metody MeSH
- HEK293 buňky MeSH
- lidé MeSH
- receptory opiátové delta metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.
- MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- chromozomální proteiny, nehistonové chemie genetika metabolismus MeSH
- difuze MeSH
- fluorescence MeSH
- fotovybělování MeSH
- FRAP metody MeSH
- konfokální mikroskopie metody MeSH
- lidé MeSH
- matematika MeSH
- molekulární modely MeSH
- myši MeSH
- reprodukovatelnost výsledků MeSH
- roztoky MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
