Another FRET technique which has been used extensively to detect proximities between fluorophores is that of hetero-FRET . Several hetero-FRET methods exist (see Fig. 3.5), and most have been applied to study GPI-anchored proteins (for a review, see ). In the early studies, Kenworthy and Edidin , when examining FRET between donor and acceptor labeled antibodies against 5'-nucleotidase (5'-NT; a GPI-AP), reported that most proteins on the surface of fixed cells were monomers. These authors overexpressed the large quantities of proteins in MDCK cells and found that FRET, as reported by the acceptor photobleaching method (measuring donor dequenching after photobleaching of acceptor), was dependent on the density of the labeled acceptors. A comparison of data with theoretical predictions for FRET in membranes ruled out any significant clustering between 5'-NT. In order to address the discrepancy between their results and those of
Varma and Mayor, Kenworthy et al. investigated FRET between FR-GPI in unfixed living cells  and failed to detect any significant clustering. These authors also examined FRET between either glycosphingolipid GM1 (as probed by the binding of fluorophore-labeled CtxB) or GM1 and GPI-AP. Although FRET was detected in each case (indicating the presence of these molecules in proximity to each other), the data ruled out any significant clustering as predicted by theoretical modeling. It is interesting to note however, that Kenworthy et al. also failed to detect any significant dimerization of CD59 as reported by Hatanaka et al. using cross-linking and biochemical (PAGE) detection . However, all of these results showing a lack of hetero-FRET detection were consistent with the predictions of hetero-FRET modeling described by Sharma et al. . Taken together, the results of these studies provide a complex picture of GPI-anchored protein organization, and also generate new tools and concepts for an understanding of the organization of "raft" components in membranes.
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