Nanostructures in Biological
Membranes
Ayman Haidar
Supervised by : Prof.I.Bernhardt
Outline
• Brief Overview about cell membrane and components
• Definition of Lipid Rafts
• Historical Overview
• Function of Lipid Rafts and Examples
• Visualization methods for Lipid Rafts
• Conclusion
Human cell membrane
•Bilayer
•Phospholipids
•Proteins
•Glycolipids
Components of a cell membrane
© Wikipedia
1. Non-raft membrane
2.
3.
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5.
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7.
8.
Lipid raft
Lipid raft associated transmembrane protein
Non-raft membrane protein
Glycosylation modifications (on glycoproteins and glycolipids)
GPI-anchored protein
Cholesterol
Glycolipid
Properties and Function
1. Phospholipids (glycerophospholipids and phosphosphingolipids)
•
Glycerophospholipids are glycerol based phopholipids and the most abondant
•
Structural component of the cell membrane and emulsifier
•
Phosphosphosphingolipids found mainly in nerve cell axons and lipid rafts
•
It is important for signal transduction
Properties and Function
2. Cholesterol
• It is a waxy steroid consisting of a polar head + bulky steroid + hydrocarbon
•It build and maintain menbrane , modulates membrane fluidity over
temperature variation
3. Glycolipids
• Its is a carbohydrate chain associalted with a phopholipids
• Recognition sites and cell attachment
Properties and Function
4. Membrane Proteins
• Integral membrane portein, peripheral membrane protein and Lipidanchored protein
• They play a role in structure and stability , cell adhesion , specific
receptors , transport and carrier
Lipid Rafts
It is the nanodomains in the membrane where there is a combination of
all previous four mentioned contents . It forms the so-called
glycolipoproteins domains
galactosyl ceramide (GalCer)
Historical Background
Singer-Nicolson 1972 :
Fluid mosaic Model
Stier & Sackmann and Klausner & Karnovsky 1970 – 1974 :
Microdomains in cell membrane
Stier & Sackmann and Israelachvili 1975:
Clusters of lipids
Karnovsky 1982
formalized the state of lipid microdomains
Simons and Ikonen 1997
Lipid Rafts
Lipid Raft‘s Propreties
• Size between 10 – 200 nm
• High concentration of cholesterol
• Enriched in sphingolipids (specially sphingomyelin)
• Resists non-ionic detergants therfore it is called DRMs(detergant resistant membrane)
• Two main type :
- Planar : more difficult to investigate due to the poor distinguishable morphological
features (they form a continuity with the plane of the membrane) , containing flotillin
protein and exists in neurones
- Caveolae : Flask shaped invagination of the plasma membrane , contains caveolin
protein and are the most observed type of lipid rafts. They exists in brain blood
microvessels , endothelial cells and different nerve cells
• Both types stays enriched of sphingolipids and cholesterol , the combination of flottilin
and caveolin has and important role in signal transduction specially for neurotrnsmittors.
As well they can be a signal inhibitor sometimes.
Lipid Raft‘s Function
They can play a role in membrane fluidity , protein movement (trafiking) , cell
to cell interaction but the main and important role stays the signal transduction
Some of these important signal transduction are :
• IgE signaling
• T-cell antigen receptor
• B cell antigen receptor
• HIV
IgE Signalling
• First discovered lipid rafts envolving signaling process
• Proofed by tritonx , fluorescence and cholesterol
T-cell antigen receptor
• More severe clustering after Lck activation
• Downregulated by CBP
HIV receptor
• Lipid rafts can provide docking
sites for opportunistic viruses
•A lipid raft 10 to 500
nanometers in diameter is the
perfect size for free-floating viral
agents such as human
immunodeficiency virus (HIV) to
bind with and infect individual
cells
•Occurs in GalCer (Galactosyl
Ceramid) riched rafts
• Cholesterol main inducer of
HIV-1
Observation and detection of lipid
rafts
They are difficult to detect due to small nanosize , membrane thickness (5nm)
and the less immobility time (always in diffusion through membrane)
Many methods to detect :
1. Normal Fluorescence microscopes using special dyes for specific proteins or
Laurdane lipophilic dye where the fluorescent react differently between
membrane phase (bulk or rafts)
2. Manipulation of cholesterol level
3. Fluorescence resonance energy transfer (FRET)
4. Atomic force microscopy (AFM)
5. Stimulated emission depletion microscopy (STED)
6. Near field scanning optical microscope (SNOM)
Fluorescence microscopy
• Fluorophores conjugated to cholera-toxin B-subunit, which binds to the raft
constituent ganglioside GM1 are often used
• Lipophilic membrane dyes are also used which either partition between
rafts and the bulk membrane, or change their fluorescent properties in
response to membrane phase (Laurdan)
Cholesterol manipulation
• Sequestration using nystatin or amphotericin
• Depletion and removal using methyl-B-cyclodextrin (MBC)
• Inhibition of cholesterol synthesis using HMG-CoA reductase inhibitors
Ex . In low cholesterol level we see a symetric growth and after some
minutes it turns as assymetric growth
FRET
• Macroscopic domain
formation in giant
unilamellar vesicles that
are composed of
cholesterol, 1,2dipalmitoylphosphatidylc
holine (DPPC) and 1,2dioleoylphosphatidylchol
ine (DOPC) and
visualized using dyes
that preferentially
partition into Lo domains
(orange) and
Ld domains (green)
• Ld liquid disorder and
Lo liquid order
SNOM
• Resolution till 30 nm
•Optical and topography image
•Need clean room
•First real images for lipid drafs in
natural cell membrane
STED
• Resolution to 4nm
• It relies on pairs of
synchronized laser pulses
Conclusions
• 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
Thank you for your attention !!!