Structural Biochemistry/Lipids/Membrane
Fluidity
The Fluid Mosaic Model
The fluid mosaic model was originally proposed by S. Jonathan Singer and Garth Nicolson in
1972. Their idea of this model was to show and describe the general structure of a biological
membrane. Biological membrane is composed of a lipid bilayer" that is essentially a twodimensional solution composed of lipids and proteins. The lipid bilayer functions as both a
solvent for integral proteins as well as a permeability barrier.
To break down each part of the name of the model: The fluidity of the model is based on the
hydrophobic components like proteins and lipids. These two components allow for the
membrane to have a fluid motion since it is not solid; it is not made of only one type of
macromolecule. The mosaic part of the name of the model is based on the fact that mosaics are
created by using different pieces to obtain an overall picture. The mosaic model consists of not
just one type of integral component but rather multiple (such as glycoproteins or phospholipids).
Because of the model being made of different pieces, it creates a mosaic, hence the name.
Proving the Fluid Mosaic Model
Freeze Fracture & Electron Micrographs
One aspect of the Fluid Mosaic Model is that membrane proteins are randomly distributed
throughout the plane of the membrane due to their mobility (lateral diffusion). This was verified
using electron microscopy to view lipid bilayers cleaved by freeze fracture.
The general outline of the technique is this:
1. Cells are frozen in liquid nitrogen.
2. Frozen cells are fractured using a knife. The fracture occurs on lines of weakness like between
the lipid bilayer of the plasma membrane.
3. Freeze Etching uses a vacuum to remove surface ice.
4. The first part of making a replica is shadowing with platinum vapor at a 45-degree angle to the
surface.
5. The next part of making a replica is evaporating a very thin layer of Carbon onto the surface at
a 90-degree angle.
6. The final replica is revealed by degrading the organic cell material away with an acid or base.
7. The Carbon-Platinum replica is then studied under an electron microscope, and the pattern of
membrane proteins is shown by the shadowed craters and bumps.
Freeze Fracture (along with metal shadowing and imaging via electron microscopy) is a
technique that can be used to visualize membrane structure and protein distribution. First, a cell
is rapidly frozen in liquid nitrogen. Then it is cleaved along the fracture plane that splits the lipid
bilayer. Separation along this plane exposes the proteins embedded in the membrane. After
fracture, the two sections are coated/shadowed with a heavy metal like platinum. Acid is used
next to dissolve the organic material, resulting in a replica of the surfaces of the sample. The
replicas are then viewed with an electron microscope. The micrographs show bumps on the
surface of the sample, which actually are transmembrane proteins since the surfaces of the
sample's halves correspond to the inner faces of the phospholipid bilayer. This confirmed that
membrane proteins are randomly dispersed throughout the phospholipid bilayer, and that there
are integral transmembrane proteins that span the entire membrane.
Example of technique and the resulting micrographs
Lateral Diffusion
Lateral diffusion refers to the lateral movement of lipids and proteins found in the membrane.
Membrane lipids and proteins are generally free to move laterally if they are not restricted by
certain interactions. Lateral diffusion is a fairly quick and spontaneous process.
Lateral diffusion can be tracked by a process called fluorescence recovery after photobleaching
(FRAP). This process is available because the use of fluorescence labeling allows the tracking of
the molecules. The cell surface will be labeled first with a chromophore, then analyzed under a
fluorescence microscope on one section (illuminated area). On this specific site, the fluoresced
molecules are destroyed by bleaching them (use of laser) and watching if they leave or enter the
illuminated area. If the molecules are mobile, it has two different states, bleached or unbleached.
If the molecule is leaving the illuminated area, this means that the molecule is bleached. If the
molecule is entering the illuminated area, this means that the molecules is unbleached. The
unbleached molecules help to increase the fluorescence intensity.
Lateral diffusion can also be measured by a complementary strategy know as fluorescence loss in
photo-bleaching (FLIP). In this technique, a small area is continuously bleached and the
fluorescent proteins are bleached as they diffuse into it. Eventually, the number of fluorescent
proteins will decrease and will result in all bleached proteins. From both FRAP and FLIP, we can
calculate the diffusion coefficient from the bleached proteins.
Although these two techniques seem promising, it has some drawbacks. One problem is that
individual movement of each protein cannot be observed because there are too many
bleached/fluorescent proteins. For example, one cannot tell whether each individual protein is
immobile or if it's only restricted to a small area, perhaps by cytoskeleton impediment. In order
to circumvent this problem, a technique known as Single-particle tracking can be used. In this
technique, an individual protein is labeled by antibodies and are colored by fluorescent dye or
small gold specks. The movement of these proteins are then recorded by video microscopy.
Using this technique allows one to observe the diffusion pathway of a protein periodically.
Transverse Diffusion
Transverse diffusion or flip-flop involves the movement of a lipid or protein from one
membrane surface to the other. Unlike lateral diffusion, transverse diffusion is a fairly slow
process due to the fact that a relatively significant amount of energy is required for flip-flopping
to occur. Most large proteins do not flip-flop due to their extensive polar regions, which are
unfavorable in the hydrophobic core of a membrane bilayer. This allows the asymmetry of
membranes to be retained for long periods, which is an important aspect of cell regulation.
In many cells, there will be protein constituents that help with the "flip-flop" process. This can be
seen through comparing the flip-flop rate of a man-made lipid bilayer" versus a natural bilayer.
The flip-flop rate of the man-made lipid bilayer is so slow that it can be considered to be idle as
compared to the natural bilayer, indicating the existence of something in the natural bilayer that
helps with process of "flipping". The flip-flop rate of phospholipids in the biological membranes
is far greater than that in the artificial lipid membranes because biological membranes have
protein constituents such as flipase and phospholipid translocases that accelerate the rate at
which phospholipids move from one side of the bilayer to the other one.
Control of Fluidity
The fluidity of membranes determines the extent to which molecules can be transported and
signals can be transducted through the membrane. Membrane fluidity is a function of its fatty
acid and cholesterol content. Fatty acid chains may be ordered and rigid or disordered and fluid
which affects the fluidity of the membrane in which they are contained. Long fatty acid chains
are able to form stronger intermolecular interactions which restrict fluidity. Bends and kinks in
the fatty acid chains formed as a result of unsaturated cis and trans double bonds may interfere
with intermolecular interactions which promotes fluidity. Membrane fluidity can therefore be
controlled by varying the number of double bonds and the length of fatty acid chains.
Meanwhile, the presence of bulky cholesterol molecules within the membrane restrict fluidity.
Cholesterol is the key regulator of membrane fluidity in animals. It is able to interact with and
form specific complexes with phospholipids that are called lipid rafts that concentrate in specific
regions of the membranes. Lipid rafts result in moderation of membrane fluidity which causes
the membranes to be less fluid while also making them less vulnerable to phase transitions.
Cholesterol is comprised of a steroid with an -OH hydroxy group conjugated at one end and a
hydrocarbon chain at the other. The rings of the steroid and the hydrocarbon chain are able to
insert themselves into the phospholipid bilayer of the membrane and participate in hydrophobic
interactions while the polar hydroxy group interacts with the polar head groups of the
surrounding phospholipids.
Melting Temperature
The extent of fluidity can be determined by the membrane's melting temperature. As heat is
increased, the membrane makes a sharp transition from a rigid state to a more fluid state. A low
melting temperature indicates the presence of fatty acids that promote fluidity while a high
melting temperature indicates the presence of fatty acids and cholesterol that restrict fluidity.
The melting temperature can also be affected by the ability of the molecule to pack themselves.
Characteristics such as saturation, double bonds (cis or trans), and length of the fatty acid chain
will affect the melting temperature. When comparing saturation versus unsaturation, the
saturated fatty acid will have a higher melting temperature because the residues will react with
each other causing the fatty acid to be in a more rigid state. The specific type of double bonds
also affect the melting temperature. Cis double bonds will have a lower melting point compared
to trans double bonds because they cannot pack themselves into a crystal as well as trans double
bonds; as a result, fats with cis double bonds more readily exit the solid phase and enter the
liquid phase. Longer chains of fatty acids will have high melting point because compared to
shorter chains, there are more bonds to break. The more bonds present, the higher the melting
point.
Other Experiments
When the fluid mosaic model was being developed, the idea that proteins could exhibit lateral
movement in a membrane was a relatively new one. One of the earliest experiments to test this
was performed by L. Frye and M. Edidin. Human cells and mouse cells were fused together and
fluorescent labeled antibodies were used to visualize whether integral membrane proteins of each
type could move among the membranes of both types in the fused cell. Rhodamine, a red
fluorescent marker was used to label the antibodies specific for human proteins and the green
marker fluorescin was used to label antibodies specific for mouse cell proteins. The newly fused
mouse/human cells were exposed to both antibodies, and the resulting binding pattern revealed
that the fused cells were not split into half red sides and half green sides, but had a intermixed
content of green and red labeled proteins/antibodies. This showed that in a short amount of time,
the integral proteins in fused cells were dispersed and therefore that membrane proteins have
rapid lateral mobility.
References
"Molecular Biology of THE CELL." Fifth edition- Alberts, Johnson, Lewis, Raff, Roberts,
Walter
Lefers, Mark. Freeze-Fracture Technique. Northwester Biology Glossary. 17 Nov 2009
http://www.biochem.northwestern.edu/holmgren/Glossary/index.html
http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Fluid_Mosaic_Model
http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Lipid_Bilayer
http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Membrane_Lipids
http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Fatty_Acids
http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Cholesterol