Lipid raft
Encyclopedia
Glycosphingolipids
Glycosphingolipids are a subtype of glycolipids containing the amino alcohol sphingosine. They include:* Cerebrosides* Gangliosides* Globosides...
and protein receptors organized in glycolipoprotein microdomains termed lipid rafts. These specialized membrane microdomains compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules, influencing membrane fluidity
Membrane fluidity
In biology, the membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane.The membrane phospholipids incorporate fatty acids of varying length and saturation...
and membrane protein
Membrane protein
A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes.-Function:...
trafficking, and regulating neurotransmission
Neurotransmission
Neurotransmission , also called synaptic transmission, is the process by which signaling molecules called neurotransmitters are released by a neuron , and bind to and activate the receptors of another neuron...
and receptor trafficking. Lipid rafts are more ordered and tightly packed than the surrounding bilayer
Bilayer
A bilayer is a double layer of closely packed atoms or molecules. The properties of bilayers are studied in condensed matter physics, often in the context of semiconductor devices, where two distinct materials are united to form junctions ....
, but float freely in the membrane bilayer.
Properties of lipid rafts
Cholesterol
Cholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
found in the surrounding bilayer. Also, lipid rafts are enriched in sphingolipid
Sphingolipid
Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named for the mythological Sphinx because of their enigmatic nature. These compounds play...
s such as sphingomyelin
Sphingomyelin
Sphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphorylcholine and ceramide...
, which is typically elevated by 50% compared to the plasma membrane. To offset the elevated sphingolipid levels, phosphatidylcholine
Phosphatidylcholine
Phosphatidylcholines are a class of phospholipids that incorporate choline as a headgroup.They are a major component of biological membranes and can be easily obtained from a variety of readily available sources such as egg yolk or soy beans from which they are mechanically extracted or chemically...
levels are decreased which results in similar choline
Choline
Choline is a water-soluble essential nutrient. It is usually grouped within the B-complex vitamins. Choline generally refers to the various quaternary ammonium salts containing the N,N,N-trimethylethanolammonium cation....
-containing lipid levels between the rafts and the surrounding plasma membrane. Cholesterol interacts preferentially, although not exclusively, with sphingolipids due to their structure and the saturation of the hydrocarbon chains. Although not all of the phospholipids within the raft are fully saturated, the hydrophobic chains of the lipids contained in the rafts are more saturated and tightly packed than the surrounding bilayer. Cholesterol is the dynamic "glue" that holds the raft together. Due to the rigid nature of the sterol group, cholesterol partitions preferentially into the lipid rafts where acyl chains of the lipids tend to be more rigid and in a less fluid state. One important property of membrane lipids is their amphipathic character. Amphipathic lipids have a polar, hydrophilic head group and a non-polar, hydrophobic region. The figure to the right shows the inverted cone-like shape of sphingomyelin and the cone-like shape of cholesterol based on the area of space occupied by the hydrophobic and hydrophilic regions. It should be noted that cholesterol has the ability to pack in between the lipids in rafts, serving as a molecular spacer and filling any voids between associated sphingolipids.
Rietveld & Simons related lipid rafts in model membranes to the immiscibility of ordered (Lo phase
Liquid ordered phase
The liquid ordered phase is a liquid crystalline phase of biological importance. It occurs in many lipid mixtures combining cholesterol with a phospholipid and/or sphingolipids e.g. sphingomyelin...
) 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
Gibbs free energy
In thermodynamics, the Gibbs free energy is a thermodynamic potential that measures the "useful" or process-initiating work obtainable from a thermodynamic system at a constant temperature and pressure...
between the two phases. Studies have shown there is a difference in thickness of the lipid rafts and the surrounding membrane which results in hydrophobic mismatch at the boundary between the two phases. This phase height mismatch has been shown to increase line tension which may lead to the formation of larger and more circular raft platforms to minimize the energetic cost of maintaining the rafts as a separate phase. Other spontaneous events, such as curvature of the membrane and fusing of small rafts into larger rafts, can also minimize line tension.
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 detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
s, 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.
History
Until 1982, it was widely accepted that phospholipidPhospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...
s and membrane protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s were randomly distributed in cell membranes, according to the Singer-Nicolson fluid mosaic model, published in 1972. However, membrane microdomains were postulated in the 1970s using biophysical approaches by Stier & Sackmann and Klausner & Karnovsky. These microdomains were attributed to the physical properties and organization of lipid mixtures by Stier & Sackmann and Israelachvili et al. In 1974, the effects of temperature on membrane behavior had led to the proposal of "clusters of lipids" in membranes and by 1975, data suggested that these clusters could be "quasicrystalline" regions within the more freely dispersed liquid crystalline lipid molecule. In 1978, X-Ray diffraction studies led to further development of the "cluster" idea defining the microdomains as "lipids in a more ordered state". Karnovsky and co-workers formalized the concept of lipid domains in membranes in 1982. Karnovsky's studies showed heterogeneity in the lifetime decay of 1,6-diphenyl-1,3,5-hexatriene, which indicated that there were multiple phases in the lipid environment of the membrane. One type of microdomain is constituted by cholesterol and sphingolipid
Sphingolipid
Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named for the mythological Sphinx because of their enigmatic nature. These compounds play...
s. They form because of the segregation of these lipids into a separate phase, demonstrated by Biltonen and Thompson and their coworkers. These microdomains (‘rafts’) were shown to exist also in cell membranes. Later, Kai Simons at the European Molecular Biology Laboratory
European Molecular Biology Laboratory
The European Molecular Biology Laboratory is a molecular biology research institution supported by 20 European countries and Australia as associate member state. EMBL was created in 1974 and is an intergovernmental organisation funded by public research money from its member states...
(EMBL) in Germany
Germany
Germany , officially the Federal Republic of Germany , is a federal parliamentary republic in Europe. The country consists of 16 states while the capital and largest city is Berlin. Germany covers an area of 357,021 km2 and has a largely temperate seasonal climate...
and Gerrit van Meer from the University of Utrecht, Netherlands refocused interest on these membrane microdomains, enriched with lipids and cholesterol, glycolipid
Glycolipid
Glycolipids are lipids with a carbohydrate attached. Their role is to provide energy and also serve as markers for cellular recognition.-Metabolism:...
s, and sphingolipid
Sphingolipid
Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named for the mythological Sphinx because of their enigmatic nature. These compounds play...
s, present in cell membranes. Subsequently, they called these microdomains, 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. At the 2006 Keystone Symposium of Lipid Rafts and Cell Function, lipid rafts were defined as "small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein interactions" In recent years, lipid raft studies have tried to address many of the key issues that cause controversy in this field, including the size and lifetime of rafts.
Other questions yet to be answered include:
- What are the effects of membrane protein levels?
- What is the physiological function of lipid rafts?
- What effect does flux of membrane lipids have on raft formation?
- What effect do diet and drugs have on lipid rafts?
- What effect do proteins located at raft boundaries have on lipid rafts?
Common Types of Lipid Rafts
Two types of lipid rafts have been proposed: planar lipid rafts (also referred to as non-caveolar, or glycolipid, rafts) and caveolae. Planar rafts are defined as being continuous with the plane of the plasma membrane (not invaginated) and by their lack of distinguishing morphological features. Caveolae, on the other hand, are flask shaped invaginations of the plasma membrane that contain caveolinCaveolin
Caveolins are a family of integral membrane proteins which are the principal components of caveolae membranes and involved in receptor-independent endocytosis. Caveolins may act as scaffolding proteins within caveolar membranes by compartmentalizing and concentrating signaling molecules...
proteins and are the most readily-observed structures in lipid rafts. Caveolins are widely expressed in the brain micro-vessels of the nervous system, endothelial cells, astrocytes, oligodendrocytes, Schwann cells, dorsal root ganglia and hippocampal neurons. Planar rafts contain flotillin proteins and are found in neurons where caveolae is absent. Both types have similar lipid composition (enriched in cholesterol and sphingolipids). Flotillin and caveolins have the ability to recruit signaling molecules into lipid rafts, thus playing an important role in neurotransmitter signal transductions. It has been proposed that these microdomains spatially organize signaling molecules to promote kinetically favorable interactions which are necessary for signal transduction. Conversely, these microdomains can also separate signaling molecules, inhibiting interactions and dampening signaling responses.
Lipid raft and signal transduction
Signal transductionSignal transduction
Signal transduction occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response...
at the cellular level refers to any process by which a cell converts one kind of signal or stimulus into another. The movement of signal or stimulus can be simple, like that associated with receptor molecules. More complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/threonine kinases. The specificity and fidelity of signal transduction are essential for cells to respond efficiently to changes in their environment. This is achieved in part by the differential localization of proteins that participate in signalling pathways. In the plasma membrane, one approach of compartmentalization utilizes lipid rafts.
One reasonable way to consider lipid rafts is that small rafts can form concentrating platforms after ligand binding activation for individual receptors. If receptor activation takes place in a lipid raft, the signalling complex is protected from non-raft enzymes such as membrane phosphatases. Overall, raft binding recruits proteins to a new micro-environment so that the phosphorylation state can be modified by local kinases and phosphatases to give downstream signalling. Lipid rafts have been found by researchers to be involved in many signal transduction processes, such as Immunoglobulin E signalling, T cell antigen receptor signalling, B cell antigen receptor signalling, EGF receptor signalling, insulin receptor signalling and so on. In order to illustrate these principles, detailed examples of signalling pathways that involve lipid rafts are described below.
Immunoglobulin E signalling
T-cell antigen receptor signaling
MHC
-Biology:*Myosin heavy chain - part of the motor protein myosin's quaternary protein structure*Major histocompatibility complex - a highly polymorphic region on chromosome 6 with genes particularly involved in immune functions-Colleges:...
) class I and class II proteins on the surface of antigen presenting cells (APCs). The CD3 and ξ- subunits contain cytoplasmic ITAM motifs.
During the signaling process, MHCs binding to TCRs brings two or more receptors together. This crosslinking, similar to IgE signaling, then recruit doubly acylated non-receptor Src-like tyrosine kinases to phosphorylate ITAM tyrosine residues. In addition to recruiting Lyn, TCR signaling also recruits Fyn. Following this procedure, ZAP-70 (which is also different with IgE signalling) binds to phosphorylated ITAMs, which leads to its own activation and LAT activation. LAT activation is the source of signal amplification. Another difference between IgE and T cell antigen receptor signalling is that Lck activation by TCR could results in more severe raft clustering thus more signal amplification. To down-regulating the signal, one possible way is the binding of cytosolic kinase Csk to the raft associated protein CBP. Csk may then suppress the Src-family kinases through phosphorylation.
B-cell antigen receptor signaling
B cell antigen receptor (BCR) is a complex between a membrane bound Ig (mIg) molecule and a disulfide-linked Igα- Igβheterodimer of two polypetides. Igαand Igβeach contains an amino acid motif, called ITAM, whose sequence is D/ExxYxxL/Ix7YxxL/I.The process of B cell antigen receptor signalling is similar to Immunoglobulin E signalling and T-cell antigen receptor signalling. It is commonly believed that other than BCR, lipid rafts play an important role in many of the cell surface events involved in B cell activation. Their functions include signaling by BCR, modulation of that signaling by co-receptors, signaling by CD40, endocytosis of antigen bound to the BCR and its routing to late endosomes to facilitate loading of antigen-derived peptides onto class II MHC molecules, routing of those peptide/MHC-II complexes to the cell surface, and their participation in antigen presentation to T cells.
Visualization of lipid rafts
One of the primary reasons for the controversy over lipid rafts has stemmed from the challenges of studying lipid rafts in living cells, which are not in thermodynamic equilibrium. Lipid rafts are small microdomains ranging from 10–200 nm in size. Due to their size being below the classical diffraction limit of a light microscope, lipid rafts have proved difficult to visualize directly. Currently synthetic membranes are studied; however, there are many drawbacks to using these membranes. First, synthetic membranes have a lower concentration of proteins compared to biomembranes. Also, it is difficult to model membrane-cytoskeletal interactions which are present in biomembranes. Other pitfalls include lack of natural asymmetry and inability to study the membranes in non-equilibrium conditions. 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 gangliosideGanglioside
Ganglioside is a molecule composed of a glycosphingolipid with one or more sialic acids linked on the sugar chain. The 60+ known gangliosides differ mainly in the position and number of NANA residues.It is a component of the cell plasma membrane that modulates cell signal transduction events...
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.
Manipulation of cholesterol is one of the most widely used techniques for studying lipid rafts. Sequestration (using filipin, nystatin or amphotericin), depletion and removal (using methyl-B-cyclodextrin) and inhibition of cholesterol synthesis (using HMG-CoA reductase inhibitors) are ways cholesterol are manipulated in lipid raft studies. These studies allow for the observations of effects on neurotransmitter signaling upon reduction of cholesterol levels.
Sharma and colleagues used combination of high resolution imaging and mathematical modeling to provide the view that raft proteins are organized into high density nanoclusters with radii ranging over 5–20 nm. Using measurements of fluorescence resonance energy transfer between the same probes (homo-FRET or fluorescence anisotropy), Sharma and colleagues reported that a fraction (20–40%) of GPI-anchored proteins are organized into high density clusters of 4–5 nm radius, each consisting of a few molecules and different GPI-anchored proteins.
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.
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), dual polarisation interferometry
Dual Polarisation Interferometry
Dual polarization interferometry is an analytical technique that can probe molecular scale layers adsorbed to the surface of a waveguide by using the evanescent wave of a laser beam confined to the waveguide...
, 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.
Other techniques used in the analysis of lipid rafts include ELISA, western blotting, and FACS.
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 1,000 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 bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
ing 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 PI(4,5)P2
Phosphatidylinositol (4,5)-bisphosphate
Phosphatidylinositol 4,5-bisphosphate or PtdInsP2, also known simply as PIP2, is a minor phospholipid component of cell membranes...
. PI(4,5)P2 plays a large role in regulating the cell’s cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
, and disrupting PI(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 PI(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.
See also
- Cell membraneCell membraneThe cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
- Membrane proteinMembrane proteinA membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes.-Function:...
s - CaveolaeCaveolaeIn biology, caveolae , which are a special type of lipid raft, are small invaginations of the plasma membrane in many vertebrate cell types, especially in endothelial cells and adipocytes....
- CholesterolCholesterolCholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
- SphingomyelinSphingomyelinSphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphorylcholine and ceramide...
External links
- "Lipid Rafts, Signalling and the Cytoskeleton" at University of EdinburghUniversity of EdinburghThe University of Edinburgh, founded in 1583, is a public research university located in Edinburgh, the capital of Scotland, and a UNESCO World Heritage Site. The university is deeply embedded in the fabric of the city, with many of the buildings in the historic Old Town belonging to the university...
- Membrane Rafts on-line lecture by Satyajit Mayor