Proximity Measurement Using HomoFRET

Homo-FRET measures the loss of fluorescence anisotropy, resulting from the FRET process. It is a relatively less well-known approach to FRET, and is also referred to as depolarization-FRET [74,75]. In this type of FRET, fluorescence emission excited by polarized light is measured for the extent of loss of fluorescence anisotropy using appropriately placed excitation and emission polarizers (Fig. 3.6). Due to a finite spread of allowed angular dependence between the donor and acceptor fluorophores, there is a significant transfer of energy between those donor and acceptor fluorophores that are not aligned to each other, and this results in an instantaneous loss of emission anisotropy. This loss can be measured in the steady state as a change in anisotropy of fluorescence emission in the presence or absence of nearby fluorophores, or alternatively in the time domain (during the lifetime of the fluorophore) as a rapid decay of emission anisotropy.

Using this technique in the steady-state measurement, Varma and Mayor [76] demonstrated the presence of lipid-dependent protein clusters on the surface of living cells. Since the extent of depolarization (or loss of anisotropy) resulting from FRET is dependent on distance (limited by R0; [74]), increasing depolarization would be seen with increasing concentration. Using this assay, Varma and Mayor showed that fluorophore-tagged Folate Receptor (FR-GPI; a model GPI-AP) is organized in clusters maintained by cholesterol levels in the membrane, whereas the same FR ecto-domain - when linked to two different transmembrane domains -showed random organization. Cholesterol depletion led to a disruption of the clusters formed by FR-GPI. Later, Sharma and coworkers showed that GFP anchored to cell membranes via a GPI-anchor (GFP-GPI) and mYFP-GPI (mYFP is monomeric version of YFP; [77]) were also organized in cholesterol-dependent sub-reso-

Gpi Anchor Plant

Fig. 3.6 Imaging set-up used to measure steady-state anisotropy. Parallel (Ii) and perpendicular (IJ fluorescence intensity images are obtained using a set of excitation and emission polarizers. The perpendicular and parallel intensity images thus obtained are processed mathematically using software to obtain anisotropy and total intensity images.

Fig. 3.6 Imaging set-up used to measure steady-state anisotropy. Parallel (Ii) and perpendicular (IJ fluorescence intensity images are obtained using a set of excitation and emission polarizers. The perpendicular and parallel intensity images thus obtained are processed mathematically using software to obtain anisotropy and total intensity images.

lution clusters, showing that mere GPI-anchoring could result into this sub-resolution clustering [78].

Although steady-state anisotropy measurement is a valuable tool for the study of sub-resolution nanometer-scale homotypic interactions of molecules in living cells, time-resolved data provide additional information that is not available from steady-state measurements. Steady-state anisotropy reports a time-averaged picture of the fluorescence anisotropy displayed by the molecules in a population. It is difficult to interpret the multiple states/possibilities that could result in this average value for the population. It is worthwhile considering the possibility of the existence of dimers (for simplicity) in a population. Steady-state anisotropy measurements would be capable of reporting homo-FRET between dimers, but they would not allow any distinction to be made between the multiple possibilities that could result in particular homo-FRET efficiency. In a situation where a homo-FRET efficiency of 50% is recorded, this could result either from 50% FRET efficiencies between all molecules, or 100% FRET efficiencies between half of the molecules. Steady-state anisotropy measurements would not be able to distinguish between the two possibilities. Time-resolved anisotropy measurements would provide the distinct decay profile for each of the above distributions, and also provide an estimate of the fraction of molecules in the clustered organization. Moreover, anisotropy decay rates associated with FRET can also be used to estimate the inter-fluorophore distances. In addition to FRET distances, time-resolved anisotropy is also capable of resolving multiple sources of depolarization arising from segmental motion or the rotation of a molecule. Hence, time-resolved anisotropy measurements provide additional information related to the fraction of molecules in cluster and intermolecular distances between molecules undergoing FRET.

Sharma and coworkers performed homo-FRET detection with time-resolved anisotropy measurements to obtain further information on the structure of GPI-AP organization [78]. Time-resolved anisotropy measurements on GFP-GPI and mYFP-GPI expressed in living cells showed that they were present in extremely high-density clusters, with ~20-40% of molecules present in the clusters and the remainder as monomers. The estimated structure of GFP-GPI clusters (with ~30% of molecules in clusters) was verified with results obtained with FR-GPI using the method of anisotropy photobleaching (for a description, see [78]). However, neither method could provide an estimate of cluster size. Sharma et al. also developed a novel tool to estimate cluster size at nanometer scale that required the measurement of homo- as well as hetero-FRET efficiencies. GPI-APs display significant homo-FRET, but hetero-FRET was not detected between GPI-APs using multiple methods of detection. In order to explain this discrepancy, Sharma et al. performed theoretical modeling of hetero-FRET efficiencies. Initially, they calculated the efficiency of hetero-FRET for variable donor:acceptor ratios, cluster sizes and fractions of molecules in clusters. The calculations showed that small clusters with a lesser fraction of molecules accounted for the lack of hetero-FRET. As expected from theoretical calculations, the formation of heptamers of GPI-APs by aerolysin toxin [79] resulted in hetero-FRET detection. This methodology provides an upper bound on the cluster size (less than four molecules). The challenge now lies in using other techniques to detect these clusters. Using this information, FCS is a likely candidate if a suitable model for the diffusion of different species can be incorporated into theoretical analyses of the correlation function.

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Responses

  • Ferdinando
    How to measure lipid raft clustering?
    6 years ago

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