Fluorescence recovery after photobleaching (FRAP) is a well-established technique which is used to study the lateral mobility and fluorescence dynamics of proteins in membranes [47,48]. After labeling of the surface molecules with a fluorescent tag, a defined micrometer-sized area on the cell membrane is photobleached (irreversible photo-destruction) within a very short time, using a focused laser beam. This is followed by an observation of the recovery of fluorescence in the bleached area (by diffusion of fluorescent molecules from neighboring regions). Two parameters may be obtained from a rigorous analysis of the data: (1) the rate(s) of recovery, which results in an estimation of a rate of diffusion; and (2) the immobile fraction. The rates of fluorescence recovery are used to estimate the diffusion constants, whereas the discrepancy between fluorescence intensity before photo-bleaching and after complete recovery of fluorescence provides the fraction of immobile molecules . Although this procedure does not throw light on the motion of individual molecules, one clear advantage of FRAP over single molecule methods is its superior statistical confidence resulting from the averaging of large number of diffusion events. Since the technique results in the destruction of fluor-ophores to achieve the measurement, there are issues regarding the damage caused by this photobleaching phenomenon. In addition, the time scales and sensitivity of the technique are limited by instrumentation, and the many different models of diffusion may be used to explain the recovery characteristics. A prior physical picture of the diffusion process is usually essential in deciphering the data [49-51].
In one of the earliest FRAP studies of DRM-associated proteins, Ishihara et al. showed that although Thy-1 exhibited a diffusion constant similar to that of labeled lipids, while up to 50% of the protein was immobile on the surface of various cell types . Hannan et al.  also showed the presence of an immobile fraction of GPI-APs on the surface of MDCK cells. When placed in perspective of recent developments in the picket-fence model, this immobile fraction would include the proteins which diffused within pickets but were restricted in mobility at larger timescales. Moreover, the results did not provide the immobile fraction of "non-raft" protein for comparison. Oliferenko et al.  performed FRAP measurements on a transmembrane-anchored lipid raft (DRM-associated) marker CD44 expressed in EpH4 cells (polarized mammary epithelial cells). These authors found that CD44 was significantly immobilized compared to a transferrin receptor (TfR), which is not present in lipid rafts (recovery after saturation being ~19% and ~50%, respectively). Cholesterol depletion and treatment with latranaculin A (an actin-disrupting agent) led to comparatively higher mobile fractions of CD44 (28% and 40% recovery respectively compared to ~19% for untreated CD44). Oliferenko et al. took these results as evidence of the presence of CD44 in lipid rafts, but the alternative possibility of CD44 being retained by pickets and fences better than TfR (perhaps through selective interaction) could not be ruled out. Cholesterol depletion and actin disruption could merely perturb pickets and fences by acting on the cytoskeleton . Shvartsman et al. investigated the diffusion of influenza HA tagged with various anchors , and found that DRM-associated wild-type and GPI-anchored forms of HA diffused more slowly than an HA mutant that was not associated with the DRMs. However, the diffusion became comparable following the depletion of cholesterol from the cells. In cells that coexpressed the wild-type and GPI-anchored forms of HA, the patching of one form by using antibodies slowed down the diffusion of the other, thus indicating the anchor- (and hence raft-) dependent interaction between the two forms.
Using FRAP, Henis and coworkers [57,58] examined the lateral diffusion constants of wild-type and activated H-Ras and K-Ras (lipid-linked small GTPases which associate with the inner leaflet of the membrane). Whilst wild-type H-Ras was found to a greater extent in DRMs, the other isoforms (wild-type K-Ras, activated H-Ras and activated K-ras) were not at all detergent-insoluble, suggesting a functional potential of the "lipid raft" association . Interestingly, these authors did not report any significant difference in the lateral mobility of any of the proteins. Only wild-type H-Ras showed an increased lateral mobility upon cholesterol depletion. Moreover, the lateral mobility of activated H-Ras and K-Ras increased with expression level in a saturable manner, whereas the lateral mobility of wildtype H-Ras was independent of its expression level. These complicated results again highlighted the lack of a precise understanding of detergent insolubility. A more comprehensive understanding of the nature of the domains that these small-molecule GTPases occupy was reviewed recently .
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