There are several indications for cell membranes being inhomogeneous fluids and for the existence of lipid-driven phase separation. One key finding was the selective co-clustering of certain membrane components and segregation from others upon application of antibodies to living cells. Co-clustering of lipids was first observed in lymphocytes, where one ganglioside species was capped with antibodies and another species was found to redistribute into the cap . It was then shown that simultaneous addition of two antibodies against apparently homogeneously distributed surface antigens could, in selected cases, lead to their co-clustering and in other cases to their segregation . These findings were explained by certain proteins residing in small raft domains that are below the light microscopic resolution in size, and others residing outside the raft domains. Upon cross-linking by antibodies the small raft domains coalesce into visible, stable clusters that contain several different raft proteins. The antigens that were previously in the non-raft environment are excluded from the coalescing domains and thus form separate clusters upon cross-linking. How these large-scale domains containing multiple raft components could be formed in a homogeneous membrane without the occurrence of phase separation is not obvious, and an alternative explanation for this phenomenon has not been put forward. Since then, two techniques have been used to directly assess liquid order in living cells. Gidwani et al. measured the steady-state anisotropy of the lipid-probe DPH-PC, which is sensitive to cholesterol-induced liquid order. With this approach, they found that approximately 40% of the plasma membrane of mast cells is in a liquid-ordered state . More recently, Gaus et al. were able to directly visualize liquid-ordered domains in living macrophages on the light microscopic level. They applied two-photon imaging of the amphiphilic dye LAURDAN, which changes its emission peak depending on the state of its lipid environment .
Other techniques have also been employed for assessing raft domains in living cells, most of them analyzing the distribution and dynamics of membrane pro teins rather than lipids. Pralle et al. measured the local diffusion of a bead attached to a single protein molecule in the plasma membrane of fibroblasts within an area smaller than 100 nm in diameter . In this way, diffusion was not hindered by cytoskeletal constraints but was supposed to be free. Proteins previously shown to be resistant to detergent extraction diffused three times slower than detergent-soluble proteins. After cholesterol depletion, the former diffused as fast as the latter. The first group of proteins was thus assumed to reside in a raft environment and to diffuse together with the whole raft entity. After destruction of this entity by cholesterol extraction the proteins behaved as if they were diffusing in a non-raft environment. From the viscous drag and from the diffusion coefficient, the size of the raft entities was calculated to be approximately 50 nm in diameter. Extrapolated from average protein and lipid densities in cell membranes, one raft entity was calculated to contain roughly 3000 lipid molecules and 10-20 proteins.
Remarkably, Prior et al. come to a very similar size for raft domains formed in the cytoplasmic leaflet of the plasma membrane using a completely different technique . They ripped plasma membrane sheets off adherent cells and labeled them with gold-coupled antibodies against H-Ras and K-Ras, supposed to reside inside and outside of raft domains, respectively. Statistical analysis of the distribution of the gold particles revealed that 35% of H-Ras labels were clustered in domains of roughly 44 nm diameter. These domains were cholesterol-dependent. Furthermore, cross-linking of GPI-anchored green fluorescent protein (GFP-GPI) in the exoplasmic leaflet resulted in co-localization of the H-Ras clusters with the formed GPI-patches, but did not change their size. However, 20% of the non-raft protein K-Ras was also found to be clustered in domains of 32 nm diameter, although these domains were cholesterol-independent. H- and K-Ras had been reported to occupy distinct domains in the plasma membrane before . Recently, single molecule imaging of H-Ras revealed cholesterol and actin dependent domains as large as 250 nm .
An often-applied technique trying to visualize raft domains in vivo is that of FRET. Hetero-FRET, which detects energy transfer between two different fluor-ophores, has not proven successful [42-44], most likely because the probability that donor and acceptor are in the same microdomain is very low. Even cross-linking one raft marker by antibodies does not lead to appreciable recruitment of others . Recently, Mayor and coworkers refined their previous analysis  using homo-FRET (i.e., energy transfer between two fluorophores of the same kind) to study clustering of GPI-anchored proteins in the plasma membrane . By measuring the anisotropy decay over time, these authors found that 20-40% of the GPI-anchored proteins are present in small complexes of two to four molecules, while the remainder is randomly distributed as monomers. The limitation of FRET measurements becomes obvious in these studies. The technique provides information about "closeness" on a very small scale (5 nm), but is not suited for visualizing bigger entities.
The fact that raft domains are difficult to visualize in vivo has led to a number of alternative explanations, mostly describing smaller entities and, most importantly, describing the formation of these entities as a protein-driven, induced event. The smallest entity was proposed by Kusumi and colleagues, who have pioneered single-particle tracking with ultra-high sampling frequencies of 40 000 Hz. The spatial resolution achieved with this frequency is 20 nm, meaning that if the domains were significantly larger and the probe resided either inside or outside the domain for several consecutive steps, then different diffusion behaviors could be observed. Since however raft and non-raft markers displayed the same diffusion characteristics, it was postulated that rafts are extremely small, namely molecular complexes of at least three membrane components, one of which comprises a saturated acyl chain or cholesterol. Stabilized raft domains accessible to diffusion measurements would only form by clustering following stimulation (for a review, see ). Anderson and Jacobson have put forward the lipid shell hypothesis, in which roughly 80 lipid molecules are supposed to surround a raft protein and form a shell of 7 nm diameter . The shells would be thermodynamically stable structures resulting from specific binding interactions between proteins and lipids, and could target the protein into larger raft-domains. How the larger raft domains form and why the raft-protein must assemble a shell of raft-lipids around it before it can enter a raft-domain remain open questions.
The size of raft domains is heavily debated and, as a consequence of the different measurements, their existence is questioned. Consensus is reached in that the proposed domain sizes of 200 nm or larger based on single-particle tracking experiments [50,51] were most likely clustered rafts, formed and stabilized by the multi-valent beads used for the tracking. Also, the 50-nm raft calculated from the viscous drag experiments by Pralle et al.  could have been a stabilized raft in which the altered dynamics due to the optical trap led to enlargement of a previously smaller structure. This leaves us with a domain size between the 5 nm derived from the FRET measurements  and the <20 nm derived from the highspeed single particle tracking studies . Better estimates will have to await the development of new methods which can finally assess the size of isolated raft domains in vivo.
In light of the co-clustering data , the visualization of distinct liquid-ordered domains in living cells , and the evidence that isolated cell membranes phase separate in vitro , it seems reasonable to assume that native cell membranes can display phase separation. One explanation for the formation of small and transient domains in the plasma membrane lies in its composition. In contrast to ternary lipid mixtures in model systems, the plasma membrane is composed of hundreds of different lipid species and, in addition to that, a variety of proteins. Viewed over a large scale, the complexity of the plasma membrane should counteract phase separation, buffer fluctuations, and in fact protect the cell against rapid phase transitions in response to small changes in the environment. If every fusion or budding event led to a phase transition, it would be difficult to prevent leakages through the bilayer and keep the membrane tight. Viewed on a smaller scale however, the picture can appear very different. Local impurities or changes in membrane composition can allow coalescence and separation of domains containing reaction partners and thus provide a regulatory principle.
81 1 Lipid Rafts, Caveolae, and Membrane Traffic 1.5
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