Fluorescence correlation spectroscopy (FCS) provides information on the diffusion of fluorescently tagged molecules by observing the correlation of fluorophore fluctuation through a small optically delimited detection volume [60-65]. The resultant fluorescence fluctuations provide an autocorrelation curve, which could be used to calculate diffusion constant of the molecules. Whilst FCS retains the statistical advantages of FRAP, it can be performed with dilute fluorophores and with relatively less laser power. This makes it more suitable for live cell studies, with lesser photo-damage and lower fluorophore concentrations, closer to that of endogenous molecules [60,63,66].
Korlach et al. used giant unilamellar vesicles (GUVs) containing various mixtures of dilauroyl phosphatidylcholine (DLPC), dipalmitoyl phosphatidylcholine (DPPC) and cholesterol. These authors imaged coexisting phases (resulting from phase separation) with confocal fluorescence microscopy using differential probe partitioning of fluorescent probes 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiI-C20) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC). The identified phases were characterized by measuring translational diffusion of DiI-C20 by FCS measurements. The probe displayed fast mobility in fluid membrane phases, and slower mobility in ordered membrane phases. Cholesterol was found to induce changes in coexisting phase domains. In binary mixtures of DLPC/cholesterol, the fluid phase of DLPC that contained a higher cholesterol content displayed slower diffusion coefficients for DiI-C20. However, by confocal fluorescence microscopy these phases appears identical.
In a similar study, Kahya et al. studied phases in GUVs prepared from ternary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), SM and cholesterol . DiI-C18 was largely excluded from the SM-rich regions, in which the raft marker ganglioside GM1 was localized when visualized with CtxB subunit. The cholesterol content was found to be critical for the phase separation into a liquid-disordered, DOPC-enriched phase exhibiting high probe mobility and a dense, liquid-ordered, SM-enriched phase. The addition of cholesterol led to increased probe mobility for the liquid-disordered, DOPC/cholesterol mixture and decreased probe mobility for the liquid-ordered, SM/cholesterol mixture. Bacia et al. per formed FCS on a raft marker GM1 probed with fluorescently labeled CtxB subunit and compared it with a non-raft marker dialkylcarbocyanine (DiI) . In homogeneous GUVs, both probes displayed slightly different diffusion (attributed to factors other than membrane composition). Both probes also showed significantly different diffusion in GUVs that contained a raft lipid mixture (unsaturated phosphatidylcholine, cholesterol and SM). CtxB-GM1 diffused significantly more slowly than DiI, consistent with its presence in liquid-ordered domains. The depletion of cholesterol by methyl-b-cyclodextrin (mbCD) resulted in an increased mobility of CtxB-GM1, consistent with the disruption of liquid-ordered domains. Similarly, CtxB-GM1 displayed extremely slow diffusion compared to DiI in rat basophilic leukemia (RBL) cells. However, there was no increase in the mobility of CtxB-GM1 by depletion of cholesterol using mbCD. In contrast, disruption of the cytoskeleton by treatment with latrunculin A resulted in a higher mobility of CtxB-GM1. The authors speculated that these results could arise if some skeleton rafts remained associated with the cytoskeleton after cholesterol depletion, but they did not conduct an experiment in which cholesterol was depleted along with cytoskeleton disruption. A further increase in mobility could have suggested a hierarchical organization of CtxB-GM1 into cholesterol- and cytoskeleton-dependent structures. Bacia et al. also showed that the SNARE proteins syntaxin and synaptobrevin, when reconstituted into GUVs, were preferentially present in liquid-ordered phases. Interestingly, Lang et al. showed that syntaxin does not co-patch with typical raft markers such as GPI-linked proteins, and does not co-fractionate with DRMs , whereas others showed that SNAREs are highly enriched in DRMs [70,71]. Although these results related to DRM association are ambiguous, they demonstrate the gap between data obtained from model membranes and from biological membranes. In an interesting follow-up study, Bacia et al. examined the role of sterol structure in phase separation in GUVs . Sterol structure was shown not only to influence phase separation but also to cause remarkable differences in the curvatures of GUVs. Whereas both cholesterol and lophenol induced positive curvature and outward budding of liquid-ordered phases, lanos-terol and cholesteryl sulfonate treatment resulted in a negative curvature and inward budding of liquid-ordered phases.
One of the major criticisms of diffusion-based studies is lack of understanding of diffusion process in membranes with complex lipid mixtures, though a recent study conducted by Hac et al. showed some progress in this direction . These authors studied diffusion in two-component binary lipid membranes as a function of composition (fraction of two lipids) and temperature. They performed Monte Carlo simulations using the thermodynamic properties of lipid mixtures (measured by calorimetry) to predict FCS autocorrelation profiles resulting from diffusion in the lipid mixtures. The predicted FCS data agreed very well with data obtained experimentally. Although biological membranes are far more complex than a two-component system, these studies at least provided some ground rules for an understanding of diffusion in biological membranes.
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