Nanostructures in Biological Membranes
Ayman Haidar
Summary SBT2
1.Introduction
All cells are surrounded by a cell membrane (also called the plasma membrane). This is a
biological membrane or biomembrane consisting of a double layer of lipids in which proteins
are located. The cell membrane keeps the components of the cell isolated from the external
environment. It also serves as the communications interface between the cell and its
environment. Biological membranes also compartmentalize cellular functions. Inside the cell,
endoplasmic reticulum, Golgi, lysosomes, vesicles and vacuoles are surrounded by a single
biological membrane. Mitochondria and the nucleus are surrounded by two biomembranes.
The cell membrane is involved in regulating the flow of materials into and out of the
cell, mediating intercellular communication and adhesion and a multitude of other functions.
The structure and functions of the cell membrane have been defined by over a half a century
of research using biochemical, physiological, cellular and molecular techniques.
The new abilities and techniques in detecting nanoscaled features opened more doors on the
nanostructure components of the cell membrane and there relation in defining the main
assembly and role of the membranes. Thus, a new concept of the “Lipid Rafts” was the main
interest of many researcher especially that it is believed that these nanodomains in the cell
membrane has the decisive role in signal transduction although the initiator of all further
signalling cascades.
In this summary, the major constituents of the biomembranes and to understand how they are
organized into a functional cell membrane. Then learning about the nanostructures in
biological membranes which are the so-called lipid rafts , their nature, function and
importance.
2.Cell membrane Components
2.1 Phospholipids
The basic structure of biomembranes is defined by their continuous components-the
phospholipids. These molecules have an important attribute that allows them to form
membranes. One part of them associates with water while the other part shuns it. As we see
this underlies the basic organization of the biological membrane. (Figure 1)
• Hydrophilic head, "likes" water polar end
• Hydrophobic tail, "hates water" non-polar chain of fatty acids
• Tail--length & number double bonds differs in different phospholipids
. Figure 1. Phospholipids structure ©Copyright 1998-2009 Danton H. O'Day
The phospholipid bilayer defines many of the physical attributes of the membrane (e.g., how
fluid it is at any temperature). It also contains lipids that are involved in cell communication
(signal transduction) as discussed in future lectures.
• Outside: more phosphatidylcholine
• Inside: more phosphatidylserine
• Glycolipids: only on outside face
Major types of membrane glycolipids and phospholipids are shown in figure 2
Figure 2. Glycolipids and Major Membrane phospholipids ©Copyright 1998-2009 Danton H. O'Day
2.2 Cholesterol
• Steroid lipid
• Flat shape: Interdigitates between phospholipids (Figure 3)
• Present in animal cell membranes , it stabilizes the membrane and control fluidity
• Absent in bacteria; most plants--Cell walls provide stability
Figure 3. Cholesterol composition and location in the cell membrane
©Copyright 1998-2009 Danton H. O'Day
2.3 Membrane Proteins
Proteins may contain a single lipid-spanning domain (single pass) or several (multipass). They
may be linked to the membrane by a glycolipid or phospholipid anchor. Proteins that are
linked to or embedded in the cell membrane may associate with other proteins (protein protein
interactions) either on the inner or outer face of the membrane. Proteins may interact directly
with lipids in the bilayer. Each of these associations will be discussed as we progress through
the course.
The figure 4 below shows the most common liaisons that occur
Figure 4. Association of proteins with the cell membrane ©Copyright 1998-2009 Danton H. O'Day
Thus there are three main categories of membrane proteins :
 Integral membrane protein : Forming carrier and channel proteins (Transporter
proteins)
 Peripheral membrane protein : Forming membrane receptors
 Lipid-anchored protein : Major role in cell adhesion
3. The Fluid Mosaic Model of the Cell Membrane
The most widely accepted model of the cell membrane is the "Fluid Mosaic Model". By this
concept the cell membrane consists of a continuous, fluid, double layer of phospholipids.
Proteins either are embedded in the bilayer or associated with either the cytoplasmic or
extracellular face. Carbohydrates are linked to the proteins (glycoproteins) or lipids
(glycolipids) only on the extracellular side. The phospholipid profiles of the cytoplasmic and
extracellular layers differ. Cholesterol, in varying amounts depending on the cell type, lies
within the membrane serving to stabilize it. (Figure 5)
Figure 5. Fuid mosaic model of the human cell membrane
4.Protein Domains in Cell Membranes
In most cells, the membrane proteins are not randomly localized but exist in complexes that
are localized to specific domains. This is an exciting area of cell biology that is growing
rapidly. For example, RACK1 (Receptor for Activated C Kinase 1) organizes many
constituents involved in cellular signaling. One of the first cell types where protein membrane
domains were identified was the sperm cell.
5. Lipid Rafts
5.1 Definition of lipid rafts
Eventhough proteins are known to exist in domains. But the membrane isn't just made up of a
continuous bilayer in which proteins and protein domains reside. There are discontinuities
within it. lipid domains or "rafts" have been shown to exist which contain difference
concentrations of certain lipids such as cholesterol and sphingolipids. These are considered to
be sites where other specific molecules group for specific functions. The preponderance of
saturated hydrocarbon chains in cell sphingolipids allows for cholesterol to be tightly
intercalated, similar to the organization of the liquid-ordered state in model membranes. The
inner leaflet is probably rich in phospholipids with saturated fatty acids and cholesterol, but its
characterization is still incomplete. It is also not clear how the inner leaflet is coupled to the
outer leaflet. One possibility is that long fatty acids of sphingolipids in the outer leaflet couple
the exoplasmic and cytoplasmic leaflets by interdigitation.Transmembrane proteins could also
stabilize this coupling. The membrane surrounding lipid rafts is more fluid, as it consists
mostly of phospholipids with unsaturated, and therefore kinked, fatty acyl chains and
cholesterol
Raft lipids are most abundant at the plasma membrane, but can also be found in the
BIOSYNTHETIC and ENDOCYTIC PATHWAYS. Whereas cholesterol is synthesized in the
endoplasmic reticulum (ER), sphingolipid synthesis and head-group modification are
completed largely in the Golgi.
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 glycolipidenriched complexes (GEMs) or DIGs or Detergent Resistant Membranes (DRMs)
5.2 Historical overview
Until 1982, it was widely accepted that phospholipids and membrane proteins 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-1,3,5hexatriene, which indicated that there were multiple phases in the lipid environment of the
membrane. One type of microdomain is constituted by cholesterol and sphingolipids. 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 (EMBL) in Germany and Gerrit van Meer from the University of Utrecht,
Netherlands refocused interest on these membrane microdomains, enriched with lipids and
cholesterol, glycolipids, and sphingolipids, 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.
5.3 Types of lipid rafts
There is two common types of lipid rafts :
The planar lipid rafts which are defined as being continuous with the plane of the plasma
membrane (not invaginated) and by their lack of distinguishing morphological features and
rich in flotillin protein. Usually they are found in neurons where the other common type of
lipid rafts “Caveolae” are not found.
Caveolae ("little caves") are the common type of lipid rafts that have small caveolin protein
molecules localized on their cytoplasmic side which are widely expressed in the brain microvessels of the nervous system, endothelial cells, astrocytes, oligodendrocytes, Schwann cells,
dorsal root ganglia and hippocampal neurons. It is likely that the accumulation of many
proteins makes the caveolae lipid rafts become evident in the electron microscope. (Figure 6)
The caveolae have been implicated in the uptake of cholesterol by endocytosis and in the
accumulation of signal transduction and other components prior to their endocytosis by
receptor mediated endocytosis. While caveolae are known to be stable, cholesterol-rich
membrane domains containing the structure-specific protein caveolin, their potentially diverse
roles in cell function are under analysis. The membrane components are formed in the golgi
and inserted into the cell membrane but much remains to be learned about their biogenesis.
The following figure shows the ultrastructural appearance of caveolae with the dark areas
representing the caveolin protein.
Figure 6. Lipid rafts in form of caveolae in the cell membrane ©Copyright 1998-2009 Danton H.
O'Day
5.4 Lipid raft size
One reason why it has been so difficult to prove that rafts exist in cells is that they are too
small to be resolved by standard light microscopy. But if raft components are crosslinked with
antibodies or lectins in living cells, then raft protein and lipid components cluster together,
and bindraft and non-raft components separate into micronsized quilt-like patches. In
fibroblasts, raft proteins rapidly diffuse in assemblies of roughly 50 nm diameter,
corresponding to about 3,500 sphingolipid molecules. The number of proteins in each raft
depends on the packing density, but is probably not more than 10–30 proteins.We do not yet
know whether individual raft proteins are randomly distributed between different rafts, or
whether they are grouped in specialized rafts. Clusters of up to 15 molecules of the same
protein have been observed within the same raft, supporting the view that some proteins are
distributed non-randomly. However, other studies indicate that such clusters may only
represent a small population. Either way, given its small size, a raft can statistically contain
only a subset of all available raft proteins. This conclusion may have profound consequences
on how signalling through rafts can be dynamically activated by raft clustering.
5.5 Lipid Rafts Function
They can play a role in membrane fluidity , protein movement (trafiking) , cell to cell
interaction but the main and important role is the signal transduction
Some of these important signal transduction are :
• IgE signaling
• T-cell antigen receptor
• B cell antigen receptor
• HIV
5.5.1 IgE signaling
Immunoglobulin E (IgE) signaling is the first convincingly demonstrated lipid rafts involving
signaling process.Evidences for this fact includes decreased solubility of Fc-epsilon receptors
(FcεR) in Triton X-100 from steady state to crosslinking state,formation of patches large
enough to be visualized by fluorescence microscopy from gangliosides and GPI-anchored
proteins,abolition of IgE signaling by surface cholesterol depletion with methyl-β
cyclodextrin and so on. This signaling pathway can be described as follows: IgE first binds to
Fc-epsilon receptors (FcεR) residing in the plasma membrane of mast cells and basophils
through its Fc segment. FcεR is a tetramer consist of one α , one β and two γ chains. It is
monomeric and binds one IgE molecule. The α chain binds IgE and the other three chains
contain immune receptor tyrosine-based activation motifs (ITAM). Then oligomeric antigens
bind to receptor-bound IgE to crosslink two or more of these receptors. This crosslinking then
recruits doubly acylated non-receptor Src-like tyrosine kinase Lyn to phosphorylate ITAMs.
After that, Syk family tyrosine kinases bind these phosphotyrosine residues of ITAMs to
initiate the signaling cascade. Syk can, in turn, activate other proteins such as LAT. Through
crosslinking LAT can recruit other proteins into the raft and further amplify the signal.
(Figure7)
Figure 7 Lipid rafts in IgE signaling ©Copyright Wikipedia
5.5.2 T-cell antigen receptor
T cell antigen receptor (TCR) is a molecule found on the surface of T lymphocytes (T cells).
It is composed of αβ-heterodimers, CD3 (γδε) complex and ξ-homodimer. The α- and βsubunits contains extracellular binding sites for peptides that are presented by the major
histocompatibility complex (MHC) 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. (Figure 8)
Figure 8 Lipid rafts as T- cell antigen receptor ©Copyright Wikipedia
5.5.3 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 polypeptides. 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.
5.5.4 HIV and lipid rafts
Figure 9. Role of lipid rafts in fusion of human immunodeficiency virus 1 (HIV-1) to CD4+ T
cells. (a) Initial binding of HIV-1 to the host CD4+ T cell. CD4 is present in microdomains enriched in
glycosphingolipids (GSLs) (e.g. GM3/Gb3) and cholesterol. The HIV-1 surface envelope glycoprotein
gp120 binds to CD4, leaving its V3 domain available for secondary interactions with raft GSLs. At
this stage, the HIV-1 transmembrane glycoprotein gp41 is still bound to gp120 in an inactive
conformation. (b) Lateral assembly of the HIV-1 fusion complex. Once bound to CD4, the viral
particle is conveyed to an appropriate co-receptor (e.g. chemokine receptors CXCR4 or CCR5 or other
G-protein-coupled seven-transmembrane-domain receptors) by the GSL raft, which moves freely in
the outer leaflet of the plasma membrane composed of other lipids such as glycerophospholipids
(GPLs). (c) End of the binding phase. Following the primary interaction with CD4 in the raft
environment, a conformational change in gp120 renders cryptic regions of the viral glycoprotein
(including the V3 domain – shown as a hinged triangle) available for secondary interactions with an
appropriate co-receptor. As seven-transmembrane domain receptors are almost flush with the cell
membrane, binding of gp120 to the co-receptor moves the viral spike close to the target membrane. In
addition, the raft begins to disperse, allowing close contact between the co-receptor and the CD4–
gp120 complex. Additional conformational changes in the HIV-1 envelope glycoprotein trimer are
necessary to unmask the fusion peptide at the N-terminus of the transmembrane glycoprotein gp41.
These conformational changes are stimulated by GSLs, which act as lipid chaperones in the raft
environment. (d) Beginning of the fusion reaction. The conformational change in gp120 is shown on
the left; the conformational change in gp41, allowing the beginning of the fusion reaction, is shown on
the right. Once the hydrophobic fusion peptide is ejected outside the viral spike, it faces a highly polar
aqueous environment and consequently penetrates into the plasma membrane of the target cell, where
it finds stabilising hydrophobic conditions. This irreversible process induces a close contact between
the viral envelope and the plasma membrane, which fuse together, allowing the entry of the
nucleocapsid in the cytoplasm of the target cell. ©Copyright Cambridge University Press
5.6 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
The table 1 below resumes different methods of observation:
Table1. Identification methods for lipid rafts © 2000 Macmillan Magazines Ltd
Additionally other methods can be used also for detection of lipid rafts such as :



Atomic force microscopy (AFM)
Stimulated emission depletion microscopy (STED)
Near field scanning optical microscope (SNOM)
5.6.1 Fluorescence resonance energy transfer FRET
A donor chromophore, initially in its electronic excited state, may transfer energy to an
acceptor chromophore (in proximity, typically less than 10 nm) through nonradiative dipole–
dipole coupling. This mechanism is termed "Förster resonance energy transfer" and is named
after the German scientist Theodor Förster. When both chromophores are fluorescent, the
term "fluorescence resonance energy transfer" is often used instead, although the energy is not
actually transferred by fluorescence. In order to avoid an erroneous interpretation of the
phenomenon that is always a nonradiative transfer of energy (even when occurring between
two fluorescent chromophores), the name "Förster resonance energy transfer" is preferred to
"fluorescence resonance energy transfer;" however, the latter enjoys common usage in
scientific literature. FRET is analogous to near field communication, in that the radius of
interaction is much smaller than the wavelength of light emitted. In the near field region, the
excited chromophore emits a virtual photon that is instantly absorbed by a receiving
chromophore. These virtual photons are undetectable, since their existence violates the
conservation of energy and momentum, and hence FRET is known as
a radiationless mechanism. Quantum electrodynamical calculations have been used to
determine that radiationless (FRET) and radiative energy transfer are the short- and longrangeasymptotes of a single unified mechanism.
Figure 10. FRET observation of lipid rafts © 2006 John F. Hancock
•
•
Macroscopic domain formation in giant unilamellar vesicles that are composed of
cholesterol, 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2dioleoylphosphatidylcholine (DOPC) and visualized using dyes that preferentially
partition into Lo domains (orange) and Ld domains (green)
Ld liquid disorder and Lo liquid order (Figure 10)
5.6.2 Near field scanning optical microscopy SNOM
Near-field scanning optical microscopy (NSOM/SNOM) is a microscopy technique for
nanostructure investigation that breaks the far field resolution limit by exploiting the
properties of evanescent waves. This is done by placing the detector very close (distance
much smaller than wavelength λ) to the specimen surface. This allows for the surface
inspection with high spatial, spectral and temporal resolving power. With this technique, the
resolution of the image is limited by the size of the detector aperture and not by the
wavelength of the illuminating light. In particular, lateral resolution of 20 nm and vertical
resolution of 2–5 nm have been demonstrated. As in optical microscopy, the contrast
mechanism can be easily adapted to study different properties, such as refractive index,
chemical structure and local stress. Dynamic properties can also be studied at a subwavelength scale using this technique. NSOM/SNOM is a form of scanning probe
microscopy. (Figure 11) In addition, by using fluorescent labels it can provide a high level of
molecular specificity SNOM detected a highly patchy distribution of some fluorescent lipid
analogues reflecting a lipid domain structure in fixed, dried cells. The sizes of these patches
were consistent with the sizes of domains implied by measurements of lateral diffusion with
fluorescence photobleaching recovery. SNOM was also used to observe clustering of
fluorescently labeled membrane receptors; clusters of platelet-derived growth factor (PDGF)
receptors down to 80-100 nm diameter were observed on glioblastoma cells, and activationdependent clustering of ErbB2 was revealed on breast tumor cells.
Figure 11. Near field scanning optical microscopy SNOM illustration © WITec, "AlphaSNOM"
5.6.3 Stimulated emission depletion microscopy (STED)
It is a fluorescence microscopy technique that uses the non-linear de-excitation of fluorescent
dyes to overcome the resolution limit imposed by diffraction with standard confocal laser
scanning microscopes and conventional far-field optical microscopes. Therefore, it is also
described as a form of microscopy. A confocal laser scanning microscope uses a focused laser
beam to illuminate a small part of the sample being observed. The laser is tuned to a
frequency that excites fluorescence from dye molecules in the sample, and light from the
small region being excited is subsequently observed by a detector. The resolution of such a
microscope is limited to the spot size to which the excitation spot can be focused. This size
depends on system parameters, but is limited by approximately half the wavelength of the
light used. Nearby structures in the focal plane with a distance smaller than about 200 nm
cannot be resolved. The resolution along the optical axis (depth resolution) is notably
worsened, around 500 nm even with objective lenses with high numerical aperture. Within the
STED microscope, the diffraction limit is surmounted by targeted strong de-excitation of dye
molecules, in effect switching them off. A resolution of up to 5.8 nm could be achieved.
(Figure 12)
Figure 12. Illustration and setup of STED http://www.mpibpc.mpg.de/groups/hell/STED.html
6. Conclusion
•
•
•
•
We still not 100% sure of the real meaning and functions of lipid rafts
Further study on blocking the attachment function may help scientists understand how
to protect the cell from pathogenic invasion specially HIV
Further detection techniques is developed in order to see more the real functions
The Space-filling model of cholesterol and sphingolipids seems to be the most
important factor in the nucleation and polymerization of such rafts
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
Ingolf Bernhardt,“ Biomembranen – Wächter des zellulären Grenzverkehrs ” Biol.
Unserer Zeit ,5/2007 (37)

Ingolf Bernhardt, Biophysik lecture , Saarland university 2010

Kai Simons & Derek Toomre, “Lipid rafts and signal transduction” , Nature Reviews
Molecular Cell Biology 1, 31-39 (October 2000)

Danton H. O'Day , “Human cell membrane lecture 1998-2009”, University of Toronto
Mississauga

D. A. Brown and E. London, “ FUNCTIONS OF LIPID RAFTS IN BIOLOGICAL
MEMBRANES ”. Annual Review of Cell and Developmental Biology ,Vol. 14: 111136 , 1998

Fantini, J.; Garmy, N.; Mahfoud, R.; Yahi, N. “Lipid rafts: structure, function and role
in HIV, Alzheimer's and prion diseases”. Expert Reviews in Molecular Medicine ,
4 (27): 1–22 , 2004

John F. Hancock et.al, “Lipid rafts: contentious only from simplistic standpoints”.
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
Christopher J. Fielding, “Lipid Rafts and Caveolae: From Membrane Biophysics to
Cell Biology”. Wiley, March 21, 2006

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of the nanoscale dynamics of membrane lipids in a living cell". Nature 457 (7233):
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
Allen, John A. “Lipid raft microdomains and neurotransmitter signalling”. Nature 8
(2007): 128-40.

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