Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education, Warsaw
Head of Department: prof. dr hab. Barbara Czarnocka
At the beginning of June 2011, there were over 770 review articles in Medline under the heading lipid rafts and about 140 under lipid rafts in disease. Thus to write a detailed article on the subject would not be particularly useful. Therefore we take this opportunity to give a prospective Reader a general view on the subject and to discuss in retrospect our published results (1-5) and some unpublished observations (6) on detergent resistant membranes, light membrane fraction, and lipid rafts.
Cell membranes are composed of non-covalently bound lipids and proteins whose weight ratio range from about 7:3 for myelin (7) to 1:4 for inner mitochondrial membrane (8). About 30% of mammalian genome are coding membrane proteins (9). From several lipid classes known to occur in eukaryotic cells (10), most of mammalian membrane lipids belong to sterols, glycerophospholipids, and sphingolipids. The only sterol in animal cell membranes is cholesterol, but glycerophospholipids and sphingolipids are represented by an over a thousand molecular species, differing in the structures of their head groups, fatty acid, and sphingosine residues (11). The progress in studies on lipidomics are likely to expand this list (12, 13). In spite of their structural diversity, all membrane lipids share a common property: they are amphiphatic i.e. have hydrophilic and hydrophobic groups or residues. When studied in Langmuir trough this property orients membrane lipids with hydrophilic groups imbedded in water while the hydrophobic parts protrude into air. At the end of 1924 Gorter and Grendel (14) compared the surface of a monolayer occupied by lipids in Langmuir trough with the surface of erythrocytes used for extraction. Through ingenuity and luck [mutually compensating mistakes (15)] they concluded that membrane lipids form a bilayer. In the bilayer model of the membrane hydrophobic residues of lipids in two layers face each other thus avoiding contact with water, while the hydrophilic groups on each side are oriented towards it. Except for archea (archeabacteria) (16) the bilayer turned out to be an universal form of a membrane structure.
It took another 10 years to present a model where not only lipids, but also proteins and their spatial relations with the membrane were considered (17). In the Danielli-Dawson model of membrane structure the lipid core was on both sides covered by a continuous layers of proteins. Later on, Robertson refined this model introducing mucoproteins on the exoplasmic and unconjugated proteins on the cytoplasmic side of the lipid bilayer membrane (18). Even as late as 1969 the Danielli-Dawson model was considered valid (19). In 1970 Frye and Eddidin (20) presented results on intermixing of surface antigens after formation of mouse-human heterokaryons. They concluded that membrane allows diffusion of surface protein antigens therefore is fluid. The other important observation on the properties of membrane proteins was discovered by Bretscher (21) that the major erythrocyte glycoprotein is not bound to but, in contradistinction to the previous models, spans the cell membrane. Based on these and their own observation Singer and Nicholson (22) presented the fluid mosaic model of cell membranes. Singer and Nicholson divided proteins into integral (transmembrane) and peripheral. In contradistinction to integral, the peripheral proteins do not span the membrane but are bound to it through electrostatic and hydrogen bonds. It was before the discovery of glycophosphatidylnositol anchored proteins (GPI-AP) of the exoplasmic layer (23) and variously lipidated proteins of the cytoplasmic layer (24, 25) which are anchored to the bilayer through hydrophobic and van der Waals interactions (26). The proteins in this model can diffuse moving freely within the fluid lipid bilayer thus the membrane is a highly dynamic structure. The model turned out to be upgradable accommodating new data (27, 28) however Singer and Nicholson did not really consider the occurrence of domains. Fluidity is not equivalent to chaos and any membrane structure or interaction that would limit it would promote formation of domains, that is the appearance of membrane areas differing from the rest of it.
Plasma membranes are asymmetric structures. Apart from strictly controlled by cells, different distribution of lipids between exoplasmic and cytoplasmic halves of a bilayer (31, 32) membranes show lateral heterogeneity that is, consist of domains. Domains differ widely in properties such as size, half life, and composition affecting their functions. Thus in polarized cells we have apical and basolateral membranes, which in turn, may contain thousands of microdomains (33). One of such microdomains are, or for non- believer, can be lipid rafts. Even though the concept of lipid domains in membranes are much older (34), the roots of the raft theory should be traced to the hypothesis of van Meer and Simons (35) explaining the preferential sorting of GPI-AP (these proteins do not have a transmembrane domain) and glycosphingolipids to the apical membrane of canine kidney cells. As proposed by these authors, GPI-AP in the Golgi apparatus, a subcellular structure where sphingolipids are synthesized (36), form domains with glycosphingolipids. These domains, stabilized by hydrogen bonds, are subsequently exocyticaly transported as a whole, to the plasma membrane. At the moment it is difficult to assess to what extent this hypothesis is universal (33) yet recently it gained support from the observations of Klemm et al. (37) who discovered immunoisolated vesicles enriched in ergosterol and sphingolipids released from trans Golgi network.
Later on two independent observations lain foundation for the raft hypothesis: the recognition of liquid ordered phase in artificial lipid membranes (38-40), and the isolation of detergent resistant membranes (41).
Without bringing in details (42), important but bewildering for a nonprofessional reader, we should consider that lipids in artificial membranes are in three forms of order or phases (43, 44). At low temperatures the acyl chains of glycerophospholipids and sphingolipids are maximally extended, packed and ordered. The membrane is in the solid ordered so, or the gel phase. At high temperature the acyl chains show unrestrained movement around C-C bonds. The membrane is in the liquid disordered ld phase. Now the molecules can move around their axis as well as in the “plane” of the membrane (43, 44). These two forms of order are separated by the main transition temperature. This temperature looks sharp and narrow for membranes made of a single phospholipid but becomes broad and poorly defined for membranes prepared from lipid mixtures containing cholesterol, thus indicating the appearance of the third phase, i.e. liquid ordered lo. The lo phase depends on cholesterol (45). The smooth and rigid structure of cholesterol, which has an “affinity” for long, saturated chains of phospholipids, locates in their vicinity thus preventing their tight packing yet maintaining, to some extent, their extended conformation (43, 44). Phospholipid molecules in lo phase have their translational mobility no more than 2-3 fold reduced when compared to ld (46). To compare, phospholipids in so phase are almost a thousand fold less mobile than in the ld phase (44). On the other hand, cholesterol is a condensing agent limiting the fluidity of phospholipids promoting both ways the lo phase (47).
Detergents are amphiphatic molecules. Due to their reversed cone shape, in water detergents occur as micelles or monomers (48). When added to cell or artificial membrane suspensions in water solutions, detergent monomers incorporate the membrane (48, 49). After a while, the concentration of detergent molecules are so high that the lipids cannot support the membrane structure and instead a mixed micelles are formed (49, 50). Since detergents, such as Triton X-100 are heavier than water, it should be possible to separate the mixed micelles of solubilized membranes from the insoluble fraction. In 1992 Brown and Rose (41) prepared at 4°C the Triton X-100 extract of epithelial cells and subjected it to density gradient centrifugation. As compared with total membrane, the detergent insoluble fraction recovered at a 5%/35% sucrose interface was enriched in GPI-AP, glycosphingolipids, sphingomyelin, and cholesterol. The GPI-AP acquired their insolubility in the detergent after leaving the Golgi apparatus (41).
Since then, DRM fraction has been isolated from all animal cells (51), plant cells containing sterols (52, 53) and recently from sterol synthesizing bacteria (54, 55). The procedure of detergent extraction of cells followed by density gradient centrifugation resulting in the separation of lipid rich detergent insoluble fraction has become widely used for, perhaps, two reasons: It is simple to perform and allows the performer to draw conclusion about basic properties of membranes and their functions.
DRMs were instrumental to the formulation by Simons and Ikonen of the raft hypothesis (56) and later, observations with this membrane fraction were frequently cited in the first review article on lipid rafts and signal transduction (57). Initially, the experiments with model membranes demonstrated that membranes in the lo phase are less detergent soluble than in the ld phase (58, 59). Thus DRMs isolated from cells could correspond to the area of the membrane in the lo phase. This convincing assumption was challenged by the observations of Heerkloz (60). He reported, that addition of Triton X-100 to a uniform membrane preparation caused separation of lipids into patches of lo and ld phases. However. on the basis of earlier experiments (61) Brown considers effects described by Heerkloz not to have greater effect (51). Likewise, Garner at al. (62) who studied the solubilization of membranes, did not detect domain formation after the addition of a detergent.
After almost 20 years from the publication of Brown and Rose (41) a few observations about DRMs seem (almost) certain. Thus phospholipids (51, 63) and gangliosides (2) with long chain, saturated fatty acid residues prefer DRMs [but compare paper by Pike et al (64)].
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