Cell membranes consist of a continuous double layer of lipid molecules in which membrane proteins are embedded. Lipids constitute about 50% of the mass of most animal cell membranes with the rest of the mass being protein. All of the lipid molecules are amphipathic in that they have a hydrophilic or polar end and a hydrophobic or nonpolar end. 

Most lipids in the CM are randomly mixed in the lipid monolayer in which they reside. The van der Waals attractive forces between neighboring fatty acid tails are not selective enough to hold these lipids together. Some lipid molecules, however, that have long and saturated fatty hydrocarbon chains (like sphingolipids) are able to be held together transiently in small microdomains or lipid rafts. Membrane proteins with unusually long transmembrane domains also accumulate in the rafts. Because the hydrocarbon chains of the lipids concentrated in lipid rafts are longer and straighter than the fatty acid chains of most membrane lipids, the rafts are thicker and thus tend to accumulate certain membrane proteins.

Some type of lipid microdomains include 1) caveolae which accumulate the protein caveolin, 2) nanodomains which are rich in cholesterol and are located in the outer leaf of the PM. 

The lipid compositions of the 2 monolayers of the lipid bilayer in many cell membranes is also very different. This lipid asymmetry has functional significance. For example, many cytosolic proteins bind to specific lipid head groups found in the cytosolic monolayer of the lipid bilayer. As an example, the enzyme protein kinase C (PKC) is activated in response to various extracellular signals and then binds to the cytosolic face of the PM where phosphatidylserine (a phospholipid discussed below) is found. As another example, when animal cells undergo programed cell death (), phosphatidylserine rapidly translocates to the extracellular monolayer which serves as a signal to induce cells like macrophages to phagocytose the dead cell. This would not occur with phosphatidylserine on the cytosolic monolayer. This translocation is accomplished with the help of scramblase. 

Membrane Lipids

There are 3 main types of membrane lipids; 1. Phosphlipids, 2. cholesterol and 3. glycolipids.

1) Phospholipids are the most abundant membrane lipids. Phospholipids are composed of glyerol linked to two fatty acids and a phosphate group. The phosphate group can have additional molecules attached, such as the positively charged choline . Phosphatidylcholine is a common component of membranes. In the fluid mosaic model, a mosaic of proteins floats in or on the fluid lipid bilayer like botas on a pond. Carbohydrate chains are often bound to the extracellular portion of these proteins forming glycoproteins. Inside the cell, actin filaments and intermediate filaments interact with membrane proteins. Outside the cell, amny animal cells have an elaborate extracellular matrix ocmposed primarily of glycoproteins. 

Phospholipds have a polar head group and 2 hydrocarbon tails which are usually fatty acids which can differ in lenght. One tail usually has one or more cis-double bonds (unsaturated) each of which creates a small kink in the tail. These bonds increase the fluidity of the cell membrane because it makes it more difficult to pack the hydrocarbon chains together. Thus the lipid bilayers containing them are thinner than bilayers formed exclusively from saturated lipids. In addition, the double bonds make it more difficult to freeze.

There are a variety of phospholipids in eucaryotic membranes which act as a type of solvent for the proteins in the membrane. Some membrane proteins can function only in the presence of specific phospholipid head groups. Some important phospholipids in the plasma membrane of cells are the following.

• phosphoglycerides are derived from glycerol. Phosphatidylserine as well as well as phosphatidylethanolamine contain a terminal primary amino groups and are found in the inner monoloayer of the lipid bilayer. Phosphatidylcholine has choline in its head group and is found in the outer monolayer. Out of the phosphoglycerides, only phosphatidylserine carries a net negative charge. 

• sphingomylin is derived from serine. As with phosphatidylcholine (above), it has choline in its head group and is thus found in the outer monolayer of the plasmid membrane.

• inositol phosphlipids have a crucial role in cell signalling. Phosphatidylinositol is concentrated in the cytosolic monolayer. A variety of lipid kinases can add phosphate groups at distinct position on its inositol ring. The phosphoyrlated inositol phospholipids then act as binding sites that recruit specific proteins from the cytosol such as the phosphatidylinositol kinase (PI 3-kinase). 

2) Cholesterol is also a major constituent in the eucaryotic cell membranes. Cholesterol is a steroid with a polar hydroxyl group (-OH). They orientate themselves in the lipid bilayer with their hydroxyl groups close to the polar head groups of phospholipids where their rigid steroid rings interact with the hydrocarbon chains closest to the polar heads on phospholipids. In this position, cholesterol makes the lipid bilayer less deformable in this region and thereby decreases the permeability to small water soluble molecules.

3) glycolipids are sugar containing lipid molecules where they are found in the outer monolayer (noncytosolic side) of the PM. Some important glycolipids include:

• Gangliosides have a net negative charge and are thought to function in cell recognition processes in which membrane bound carboyhdrate binding proteins called lectins binds to the sugar groups on glycolipids in cell-cell adhesion. Some glycolipids provide entry points for bacterial toxins. For example, the ganglioside GM1 acts as a cell surface receptor for the toxin that causes cholera which leads to an increase in cAMP which in turn causes a large efflux of Na+ and water in the intestine.

• cerebrosides

Membrane Vesicles

Membrane vesicles are essentially spherical vesicles, generally less than 130 nm in diameter, composed of a lip bilayer containing a cytosolic fraction. Particular membrane vesicles are more specifically produced by cells, from intracellular compartments through fusion with the plasmic membrane of a cell, resulting in their release in biological fluids or in th supernatant of cells in culture.

Exosomes: are between about 300-120 nm in diameters and advantageously carry membrane proteins, particularly major histocompatility complex proteins and other others which directly or indirectly participate in antigen presentation. Depending on their origin, exosomes may also have proteins such as MHC I, MHC II, CD63, CD81 and/or HSP70 and have no endoplasmic reticulum or Golgi apparatus. Exosomes are essentially void of nucleic acids such as DNA or RNA. 

Exosome release has been demonstrated from different cell types in varied physiological contexts. For example, B lymphocytes have been shown to release exosomes carrying class II MHC which play a role in antigenic presentation. Similarly, DCs produce exosomes (i.e., dexosomes, Dex) and play a role in immune response mediation, particularly in cytotoxic T lymphocyte stimulation. Tumor cells have also been shown to secrete specific exosomes (i.e., texosomes, Tex) carrying tumor antigens and capable of presenting these antigen or transmitting them to APCs. 

Lamparski (WO 01/82958) discloses methods of preparing membrane visciles by obtaining a population of cells such as DCs, optionally sensitizing the DCs to antigen(s) culturing the DCs under conditions which allow them to release membrane vesicles, clarificaiton of the culture supernatant, concentraiton of the supernatant, diafiltraiton of the concentrated supernatant, isolation of the memrane vesicles using density cusion centrifugation, contacting the membrane viscles with a peptide to produce peptide loaded membrane vesicles , optionally a diafiltration buffer exchange step and sterile filtraiton of these membrane vesicles. 

Membrane Proteins

Membrane proteins can be purified with detergents which are small amphipathic molecuels that tend to form micelles in water. When mixed with membranes, the hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, thereby displacing the lipid molecules. Since the other end of the detergent is polar, this binding tends to bring the membrane proteins into solution as detergent-protein complexes. The polar ends of detergents can be either charged (ionic), as in sodiuim dodecyl sulfate (SDS) or uncharged as in triton detergents. With strong ionic detergents like SDS, even the most hydrophobic membrane proteins can be solubilized. Many hydrophobic membrane proteins can be solubilized and then purified in an active form by the use of mild detergents like triton X-100. Membrane proteins can be broadly classified as follows:

• Transmembrane proteins are amphipathic proteins that extend through the entire lipid bilayer of the PM. Their hydrophobic regions interact with the hydrophobic tails of the lipids while their hydrophilic regions are exposed to water on either side of the membrane.  In most transmembrane proteins the polypeptide chain crosses the lipid bilayer in an  because hydrogen bonding between the peptide bonds (which are polar) of these proteins is maximized if the polypeptide chain forms an alpha helix as it crosses the bilayer.

• Proteins containing about 20-30 amino acids with a high degree of hydrophobicity are long enough to span a lipid bilayer as an alpha helix and can often be identified by means of a hydropathy plot. In single pass transmembrane proteins, the polypeptide crosses the bilayer only once whereas in multipass transmembrane proteins, the polypeptide chain crosses multiple times. One such multipass transmembrane protein that can also satisfy the hydrogen bonding requirements of the polypeptide is the so called B barrel. In this conformation, the multiple transmembrane strands of the protein are arranged as a B sheet in the form of a closed barrel. 

  Most transmembrane proteins in animal cells are glycosylated. The sugar residues are added in the lumen of the ER and Golgi apparatus. Because of this, the oligosaccharide chains are always present on the noncytosolic (inside the cell) side of the PM. Disulfide bonds also form between cysteine residues on the noncytosolic side of the PM where they stablize the protein. Such bonds can not form on the cytosolic side due to the reducing environment of the cytosol of the cell.

  The 3 major transmembrane proteins responsible for cellular communication are 1) , 2) and 3) .

  • peripheral membrane proteins do not extend into the lipid bilayer but are rather bound to the face of the membrane by noncovalent interactions with other membrane proteins. 

  • GPI anchored proteins are linked via an oligosaccharid linker to phosphatidylinositol in the noncytosolic bilayer. GPI linked proteins are made as single pass transmembrane proteins in the ER. While still in the ER, the transmembrane segment of the protein is cleaved off and a glycosylphosphatidylinositol (GPI) anchor is added.