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Apparent from the plots of Figure 4 is a time-dependent change in the FRET efficiency measured for the cell displayed in Figure 3.This was likely due to the binding of calcium to the calmodulin-M13 moiety of the Cameleon sensor and subsequent change in configuration of the Cameleon sensor. Stimulation of the M1-expressing cells with carbachol is known to lead to an increase in inositol phosphates, which in turn releases intracellular Ca2+ stores (7). The Ca2+- induced configuration change in the Cameleon biosensor leads to the donor and acceptor being brought closer to each other, and hence produces the increase in FRET efficiency.
Careful inspection of the shape of the plot of Figure 4B allows elucidation of the cellular events leading to the observed changes in Ca2+ concentration. The initial steep increase in FRET occurring over a time period of thirty seconds signifies the point when the carbachol concentration in the exterior near to this particular cell was high enough to initiate a cascade of events which eventually leads to the opening of the internal calcium stores within the cell. Following this rapid increase in average FRET efficiency is a slight decrease to a prolonged plateau of FRET efficiency values. This plateau state is indicative of the opening of calcium release activated channels following the depletion of the internal calcium stores (7).
Stoichiometry and structure of a G protein-coupled receptor oligomerYeast cells (Saccharomyces cerevisiae) were genetically engineered to express the pheromone receptor ste2 fused to either GFP2 (donor) or YFP (acceptor), as described previously (5,8). Yeast cell colonies selected for the two plasmids of interest were placed on the surface of a microscope slide and imaged using OptiMiS. Typical images obtained with this system are shown in Figure 5, while the corresponding histogram of FRET efficiencies is pictured in Figure 6. Notably, this histogram differs in shape and width when compared to similarly constructed histograms of the CHO cells expressing oligomeric constructs characterized by a single FRET efficiency (see Figure 2). Specifically, multiple peaks are observed in the histogram of Figure 6, suggesting the population of ste2 oligomers consists of sub-species characterized by different FRET efficiency values. These subpopulations occur in oligomer complexes of size greater than two due to the naturally occurring permutations of the ratio and placement of the donor- and acceptor-tagged protomers in the oligomeric complex. The measured data (blue circles) displayed in Figure 6 are simulated with a model of a rhombus-shaped tetramer, which predicts five Gaussian functions (red solid line). Each Gaussian corresponds to a certain combination of number of donors to acceptors in the cell (9). This result is consistent with previously published reports (5).
Conclusion
OptiMiS gives researchers the ability to finely discern, through a single scan, the FRET efficiency of dimeric or oligomeric molecular complexes within single cells. This level of FRET efficiency resolution is highly valuable in protein complex stoichiometry and configuration studies, as well as when using sensors based on FRET. In addition, OptiMiS may be used for techniques which have not been illustrated in this short note, e.g. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS). Because OptiMiS acquires all the necessary spectral information in a single excitation scan of the sample, the acquisition time can be reduced such that changes in FRET efficiency, which may be occurring over time due to changes to the structure of the molecular complex, can be accurately monitored.
Address correspondence to J. Oliver (
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