Max-Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307, Dresden, Germany
* Author for correspondence (e-mail: simons{at}mpi-cbg.de)
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Introduction |
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Rafts are liquid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. The tighter packing is due to the saturated hydrocarbon chains in raft sphingolipids and phospholipids compared with the unsaturated fatty acids of phospholipids in the non-raft phase (Simons and Vaz, 2004). Recent studies have suggested that an equivalent domain organisation could be present in the cytoplasmic leaflet as well. However, the properties of this inner leaflet have not been adequately defined (Parton and Richards, 2003
). Theoretical considerations predict that a liquid-ordered packing of the outer leaflet leads to a more ordered packing also of the inner leaflet (Israelachvili, 1973
).
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Although a large fraction of the cell surface proteins are found in the liquid disordered regions, some proteins preferentially partition into the ordered raft domains. Typical examples include the glycosylphosphatidylinositol (GPI)-anchored proteins, which are attached to the outer leaflet of the membrane via the GPI anchor (Chatterjee and Mayor, 2001); the Src-family tyrosine kinases (e.g. Lck, Fyn and Lyn), which are anchored to the inner leaflet via their dual acylation modification (Simons and Toomre, 2000
); palmitoylated and myristoylated proteins such as flotillins (Rajendran et al., 2003
); cholesterol-binding proteins such as caveolins (Kurzchalia and Parton, 1999
) and hedgehog (Karpen et al., 2001
); heterotrimeric G proteins; and phospholipid-binding proteins such as annexins (Babiychuk et al., 2002
). One morphologically identifiable raft structure is the caveola (Kurzchalia and Parton, 1999
). Caveolae are flask-shaped membrane invaginations found in the plasma membranes of several types of cell enriched in their scaffolding proteins, caveolins. Overexpression of these proteins in cells lacking caveolae, such as lymphocytes and neuronal cells, can induce the formation of caveolae (Fra et al., 1995
), and targeted disruption of caveolin 1 in mice leads to the disappearance of morphologically recognisable caveolae. Flotillins are non-caveolar proteins that localise to microdomains and probably function as raft organisers. Annexins have also been reported to organise rafts in a calcium-dependent fashion.
Rafts are dynamic and this means that both proteins and lipids can move in and out of raft domains with different partitioning kinetics. Despite much evidence supporting the existence of raft domains, the size and the functions of these domains are debated (Edidin, 2003). The controversy mainly arises because these domains are too small to be optically resolved. However, recent advances in imaging are now providing insights into their behaviour (Gaus et al., 2003
; Parton and Richards, 2003
; Pralle et al., 2000
). Depending on the time-resolution of the technique used, different properties can be revealed (Kusumi et al., 2004
).
The first method to biochemically define lipid rafts was based on the resistance of lipid rafts to extraction by Triton X-100 at 4°C (Brown and Rose, 1992). These DRM fractions are aggregates of raft domains and thus do not represent the native state of lipid rafts in cell membranes (Munro, 2003
). One confusion in this field was caused by the equation of lipid rafts to caveolae (Anderson, 1998
). Caveolins are clearly a part of DRMs in cells that express these proteins but form a subclass of rafts (as explained above). A number of new methods are being introduced to study rafts in cells and this field needs better methodology if we are to come to grips with these elusive membrane domains.
At steady state, rafts are too small to engage in raft-associated processes. Whatever their size is, researchers agree that these domains contain only a few proteins. To engage in membrane function, they usually have to cluster together. There is increasing evidence that the outer leaflet domains and the inner leaflet domains are coupled in raft clusters (Gri et al., 2004). Raft clustering can be accomplished from both sides of the plasma membrane. Antibodies, antigens or raft-lipid-binding proteins such as cholera toxin B, cluster rafts on the extracellular side of the membrane whereas raft-clustering proteins such annexins, flotillins or other scaffolding proteins could serve as clustering agents for the rafts in the cytoplasmic leaflet. Clustered rafts can sequester specific sets of signalling and other proteins and could serve as platforms to execute functions in membrane trafficking, signalling and polarisation (Simons and Toomre, 2000
; Harder and Engelhardt, 2004
). We review some examples below, starting with membrane trafficking, in which rafts could play an important role as sorting platforms at various stages of the endo- and exo-cytic pathways.
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Endocytosis |
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Non-caveolar internalisation routes of raft proteins
Certain raft-associated proteins have been shown to be endocytosed by clathrin-mediated endocytosis the other major mode of internalisation although this pathway mostly excludes lipid rafts (Nichols, 2003). It is possible that strong endocytic signals trap proteins into clathrin-coated pits and thus enable the proteins to bypass raft-mediated internalisation (Stoddart et al., 2002
). GPI-anchored proteins in particular can present complicated scenarios. For example, GPI-anchored CD14 is sorted in a cell-type-specific fashion. While CD14 is sorted to recycling endosomes in CHO cells, it is routed to late endosomes in BHK fibroblasts (Fivaz et al., 2002
).
In addition, there is evidence for other raft-mediated routes of internalisation (Lamaze et al., 2001; Sabharanjak et al., 2002
). Sabhanranjak et al., have described a novel raft-dependent pathway in which native GPI-anchored proteins are internalised to recycling endosomes bypassing the early sorting endosomes but via a newly identified organelle called the GPI-anchored protein enriched early endosomal compartment (GEEC). This compartment has been shown to be devoid of caveolins but accumulates the fluid-phase marker dextran along with the folate receptor. Internalisation to GEECs depends on Cdc42, a Rho GTPase, but neither clathrin nor dynamin is involved. The GPI anchor has been shown to be a GEEC-targeting signal: a transmembrane equivalent fails to accumulate in the compartment indicating that GEEC-mediated internalisation is a key endocytic pathway for non-crosslinked GPI-anchored proteins.
Interleukin receptor-2 (IL-2R), an essential lymphocyte growth factor has by contrast been shown to be constitutively associated with lipid rafts and uses a non-clathrin-mediated process for internalisation (Lamaze et al., 2001). Its internalisation process depends on dynamin activity but is independent of caveolae. The reason for several distinct raft-mediated endocytic pathways is not yet clear. It is plausible that distinct lipid raft domains, differing not only in their lipid composition but also in the nature of the proteins partitioned into these domains, employ distinct mechanisms.
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Sorting in polarised epithelial cells |
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Rafts could also play an important role in the formation of transport carriers. Domain-induced budding could provide the driving force for the formation of apical containers. Such a mechanism is proposed to involve outward bending of raft clusters and fission at the domain boundary (Schuck and Simons, 2004).
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Role of rafts in virus budding |
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HIV employs a different mechanism to exit the host cell, using the endosomal sorting complexes required for transport (ESCRT) machinery normally responsible for the formation of the internal vesicles in multivesicular bodies (MVBs) for assembly of its envelope. The site of assembly could be either the plasma membrane or MVBs (von Schwedler et al., 2003). Nevertheless, it has been shown that raft lipids play a role in this process (Aloia et al., 1988
). Most importantly, the HIV envelope is enriched in raft lipids, and both entry and exit of the virus is dependent on functional rafts.
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Rafts in immune receptor signalling |
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When T cells recognise the antigen presented on the surface of antigenpresenting cells (APCs), polarisation of lipid rafts and raft-associated proteins occurs through their association with the immunological synapse, where the T cell contacts the APC (Burack et al., 2002). In addition, T-lymphoblasts exposed to migratory signals develop polarised domains at the actin-rich leading edge and at the trailing edge. Recent studies show that one ganglioside, GM1, localises to the uropods whereas another, GM3, segregates to the leading edge. Two different raft clusters, GM1-rafts and GM3-rafts, containing different subsets of raft-associated proteins therefore become dynamically segregated during cell polarisation (Gomez-Mouton et al., 2004
). Lipids rafts thus appear to play a crucial role at the interface between signalling, membrane trafficking and cell polarisation.
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Acknowledgments |
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References |
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