Raft Domains are Clustered to Exert their Function

While the steady-state existence, size and shape of liquid-ordered domains in cells remains the subject of debate, agreement has been reached on the fact that raft domains coalesce upon cross-linking to form signaling and possibly also sorting platforms [53-55]. Cross-linking is achieved by multivalent ligands binding to surface receptors or by cytoplasmic scaffolding proteins. The initial cross-linking event is thought to increase the number of contact sites between raft proteins and lipids, which leads to a potentiation of the formerly weak interactions. The previously small raft domains coalesce and form large, more stable entities. It is the clustered state in which rafts are accessible to microscopy.

Cross-linking of raft antigens not only leads to co-clustering of raft components within one leaflet, but also influences the organization of the opposing monolayer. Cross-linking of the exoplasmic GPI-anchored PLAP led to partial co-clustering of the src-kinase fyn in the cytoplasmic leaflet of Jurkat cells [35, 56]. Cross-correlation analysis revealed co-distribution of an inner leaflet raft protein with FceRI transmembrane receptors that were cross-linked by binding of their multivalent ligand IgE, as well as with antibody cross-linked raft markers of the exoplasmic leaflet, such as the GPI-anchored protein Thy-1 or the ganglioside GDib [57]. The finding that clustering not only leads to lateral coalescence of small raft domains in the exoplasmic leaflet, but also in the cytoplasmic leaflet, strengthens the hypothesis that clustered raft domains provide a platform for bringing together signaling complexes and propagating signals into the cell (reviewed in [58]). Interestingly, also in symmetric model bilayers, liquid-ordered domains have always been observed to coincide in both leaflets [20, 59]. How the connection of the inner leaflet and the outer leaflet is achieved, remains an open question. Interdigitation of the often long fatty acid chains of glycosphingolipids has been proposed to enforce a higher order also in the cytoplasmic leaflet. Alternatively, or additionally, transmembrane proteins could mediate transbilayer coupling.

Many signaling processes have been proposed to depend on the clustering of raft domains [60,61] (see also Chapter 7), the T-cell synapse being the prime example [62,63]. According to a recent study by Douglass et al., the initial stage of signaling complex assembly does not require rafts but is rather dependent on proteinprotein interactions [64]. Studies by Magee et al., on the other hand, have shown that raft clustering independent of protein-protein interactions can activate signaling pathways downstream of the T-cell receptor [65]. These authors observed that incubating T cells at 0 °C leads to coalescence of raft components into visible domains on the plasma membrane. At the same time, chilling activates the signaling cascade, leading to increased tyrosine phosphorylation and ERK activation. The cold-induced, protein-independent coalescence of raft domains is a clear indicator for a phase separation phenomenon, since it is well established that the phase-separated domains are larger at lower temperature and fragment at higher temperature due to the increase in Brownian motion [66]. However, it is not yet clear which role this raft coalescence would play in T-cell signaling under physiological conditions.

The formation of large, clustered raft domains is easiest imagined to occur by coalescence of pre-existing, small rafts. However, a recent study on model membranes of different compositions argued that phase separation can be induced by cross-linking one component in a previously homogeneous membrane [67]. GUVs composed of PC, SM and cholesterol exhibit phase separation into a liquid-ordered and a liquid-disordered phase, depending on the ratio of the components. When a small amount of the ganglioside GM1 is included in the vesicles, its cross-linking with the pentavalent cholera toxin B subunit leads to coalescence of the GM1-containing phase into larger, visible domains. Hammond et al. showed that domains can not only be formed at GUV compositions that displayed phase separation prior to clustering, but also at compositions very close to the phase transition boundary in which no previous phase separation was detected [67]. The local increase in GM1 concentration following the cross-linking might have been enough to cross the boundary and cause the membrane to phase separate.

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