Lipid raft

Overview
A lipid raft is a cholesterol-enriched microdomain in cell membranes. Since 1972, it has been believed that, in cell membranes, phospholipids and membrane proteins are ubiquitously distributed according to a fluid mosaic model. However, in 1988, Kai Simons at the European Molecular Biology Laboratory (EMBL) in Germany and Gerrit van Meer from the University of Utrecht, Netherlands suggested the novel idea that there exist microdomains, which are enriched with many kinds of lipids such as cholesterol, glycolipids, and sphingolipids, present in cell membranes. This was the first time these microdomains were called "lipid rafts". The original concept of rafts was used as an explanation for the transport of cholesterol from the trans Golgi network to the plasma membrane. The idea was more formally developed in 1997 by Simons and Ikonen.

Properties of lipid rafts
Rietveld & Simons related lipid rafts in model membranes to the immiscibility of ordered (Lo phase) and disordered Ld or Lα phase) liquid phases. The cause of this immiscibility is uncertain, but the immiscibility is thought to minimize the free energy between the two phases.

By one early definition of lipid rafts, lipid rafts differ from the rest of the plasma membrane. In fact, researchers have hypothesized that the lipid rafts can be extracted from a plasma membrane. The extraction would take advantage of lipid raft resistance to non-ionic detergents, such as Triton X-100 or Brij-98 at low temperatures (e.g., 4°C). When such a detergent is added to cells, the fluid membrane will dissolve while the lipid rafts may remain intact and could be extracted.

Because of their composition and detergent resistance, lipid rafts are also called detergent-insoluble glycolipid-enriched complexes (GEMs) or DIGs or Detergent Resistant Membranes (DRMs). However the validity of the detergent resistance methodology of membranes has recently been called into question due to ambiguities in the lipids and proteins recovered and the observation that they can also cause solid areas to form where there were none previously.

Examples
Certain proteins associated with cellular signaling processes have been shown to associate with lipid rafts. Proteins that have shown association to the lipid rafts include glycosylphosphatidylinositol (GPI)-anchored proteins, doubly-acylated tyrosine kinases of the Src family, and transmembrane proteins. This association can at least be partially contributed to the acylated, saturated tails of both the tyrosine kinases and the GPI-anchored proteins, which matches the properties of sphingolipids more so than the rest of the membrane (Simons & Ikonen, 1997). While these proteins tend to continuously be present in lipid rafts, there are others that associate with lipid rafts only when the protein is activated. Some examples of these include, but are not limited to, B cell receptors (BCRs), T cell receptors (TCRs), PAG, and an enzyme called CD39.

Other proteins are excluded from rafts, such as transferrin-receptor and a member of the Ras family. Typically the inclusion or exclusion of proteins is determined by whether or not they are found in membrane fragments extracted using Triton - the DRM definition of a raft.

Researchers have tested the presence and importance of lipid rafts in cellular signaling by first understanding the initial signaling processes, and then disrupting the lipid rafts at which point they observe any changes in cellular function. Lipid rafts are typically disrupted by removing the cholesterol from the membrane, using systems such as cyclodextrin.

In normal B cells, when the cell encounters an antigen, the BCR shifts into a lipid raft domain and then relays a signal that causes the cell to proliferate into plasma cells and produce antibodies. However, when the cholesterol was depleted from B lymphocytes, presumably destroying lipid rafts, the BCRs were no longer able to relay the signal that they had encountered an antigen, and no antibodies were produced. In a similar fashion, when rafts were depleted in T lymphocytes, the TCRs lost their ability to relay signals due to antigen attachment as well. Lipid raft depletion also affected the function of CD39 an enzyme that plays a role in platelet aggregation.

Rafts have been implicated in a number of other processes and systems both physiological and pathological. These include cell signalling, molecular trafficking, the function of the immune, vasular, digestive and reproductive systems. The pathogenesis of diseases such as HIV (viral), Salmonella (bacterial) and malaria (eukaryotic) has been linked to the role of rafts. Typically this involves the 'hi-jacking' of the host cell raft function by the pathogen for it's own purposes, e.g. to gain access to the interior of a host cell.

Visualization of lipid rafts
Due to their size being below the classical diffraction limit of the light microscope, lipid rafts have proved difficult to visualize directly. Despite this, fluorescence microscopy is used extensively in the field. For example, fluorophores conjugated to cholera-toxin B-subunit, which binds to the raft constituent ganglioside GM1 is used extensively. Also used are lipophilic membrane dyes which either partition between rafts and the bulk membrane, or change their fluorescent properties in response to membrane phase. Laurdan is one of the prime examples of such a dye. Rafts may also be labeled by genetic expression of fluorescent fusion proteins such as Lck-GFP.

To combat the problems of small size and dynamic nature, single particle and molecule tracking using cooled, sensitive CCD cameras and total internal reflection (TIRF) microscopy is coming to prominence. This allows information of the diffusivity of particles in the membrane to be extracted as well as revealing membrane corrals, barriers and sites of confinement. The Kusumi lab are some of the leaders in this field of raft study.

Other optical techniques are also used: Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS/FCCS) can be used to gain information of fluorophore mobility in the membrane, Fluorescence Resonance Energy Transfer (FRET) can detect when fluorophores are in close proximity and optical tweezer techniques can give information on membrane viscosity.

Also used are atomic force microscopy (AFM), Scanning Ion Conductance Microscopy (SICM), Nuclear Magnetic Resonance (NMR) although fluorescence microscopy remains the dominant technique. In the future it is hoped that super-resolution microscopy such as Stimulated Emission Depletion (STED) or various forms of structured illumination microscopy may overcome the problems imposed by the diffraction limit.

Controversy about lipid rafts
The role of rafts in cellular signaling, trafficking, and structure has yet to be determined despite many experiments involving several different methods.

Arguments against the existence of lipid rafts include the following:


 * First, a line tension should exist between the Lα and Lo phases. This line has been seen in model membranes, but has not been readily observed in cell systems.


 * Second, there is no consensus on lipid raft size, which has been reported anywhere between 1 and 1000 nanometres.


 * Third, the time scale of lipid raft existence is unknown. If lipid rafts exist, they may only occur on a time scale that is irrelevant to biological processes.


 * Fourth, the entire membrane may exist in the Lo phase.

A first rebuttal to this point suggests that the Lo phase of the rafts is more tightly packed due to the intermolecular hydrogen bonding exhibited between sphingolipids and cholesterol that is not seen elsewhere.

A second argument questions the effectiveness of the experimental design when disrupting lipid rafts. Pike and Miller discuss potential pitfalls of using cholesterol depletion to determine lipid raft function. They noted that most researchers were using acute methods of cholesterol depletion, which disrupt the rafts, but also disrupt another lipid known as PIP(4,5)P2. PIP(4,5)P2 plays a large role in regulating the cell’s cytoskeleton, and disrupting PIP(4,5)P2 causes many of the same results as this type of cholesterol depletion, including lateral diffusion of the proteins in the membrane. Because the methods disrupt both rafts and PIP(4,5)P2, Kwik et al concluded that loss of a particular cellular function after cholesterol depletion cannot necessarily be attributed solely to lipid raft disruption, as other processes independent of rafts may also be affected. Finally, while lipid rafts are believed to be connected in some way to proteins, Edidin argues that proteins attract the lipids in the raft by interactions of proteins with the acyl chains on the lipids, and not the other way around.