The Plasmamembrane
and Lipid Rafts
Lecture by Dr. Dirk Lang
Dept. of Human Biology
UCT Medical School
Room 6.10.1
Phone: 406-6419
E-Mail: DIRK.LANG@UCT.AC.ZA
08/2007
Three Classes of Lipids
Build the Biomembrane
1. Phosphogylcerides
-
Polar head group attached to
the phosphate, amphipathic
-
Phosphoglycerides are
classified according to the
hydrophilic head group:
•
Phosphatidylcholine
•
Phosphatidylethanolamine
•
Phosphatidylserine
•
Phosphatidylinositol
Three Classes of Lipids
Build the Biomembrane
2. Sphingolipids
(e.g. sphingomyelin)
-
Amphipathic.
-
Closely resembles
phosphatidylcholine.
-
Can form mixed bilayers.
Three Classes of Lipids
Build the Biomembrane
3. Steroids
(e.g. cholesterol)
-
Amphipathic, because of
the OH group.
-
Cannot form its own
bilayer.
-
Can & does particpate in
phospholipid bilayers.
Lipid Molecules in the
bilayer are mobile …
 Rotationally – they can
spin.
 Laterally – Diffuse
horizontally in the
membrane.
 The bilayer is viscous,
like olive oil –
100X that of water.
 In an artificial lipid
bilayer, the rate of
diffusion is 1 μm/sec
(length of animal cell in
20 sec.)
Lipid Rafts:
Previously: “Fluid Mosaic Model” of
plasmamembrane – free lateral diffusion of
membrane lipids and proteins
However, labelling experiments indicate that
different lipids (phospholipids vs. cholesterol
and sphingolipids) segregate in the membrane,
and restrict lateral diffusion of certain proteins
Fluorescent lipids segregate in patches
(microdomains) in model membranes
Some proteins are associated with rafts:
e.g. cell surface receptors and signalling proteins
Some components of lipid cafts:
Why are lipid rafts of interest?
Many components of signalling pathways (e.g. cell
surface receptors, G-proteins, kinases) appear
clustered or enriched in rafts:
Do the rafts form centres of signal
transduction, where the individual components
can interact efficiently?
Do different rafts function to spatially
separate individual signalling pathways?
Clustering and endocytosis of cell surface
proteins through rafts?
Possible roles of rafts in vesicle traffic:
3) Membrane fusion
and exocytosis?
2) Vesicle transport
and interaction with
cytoskeleton?
1) Vesicle budding
and protein sorting?
How to analyse lipid rafts?
1) Fractionation of cells and isolation of
detergent-resistant membrane (DRM) fraction
2) Non-disruptive microscopy methods:
Single particle tracking
Colocalisation and interaction studies
After cholesterol
depletion:
No more DRM
fraction, but still
raft-like
complexes in cell
membrane
Why is the raft concept controversial?
What are Caveolae?
Caveolae (Latin: “little caves”) are structurally
distinct microdomains of the plasmamembrane;
they contain the structural protein caveolin
Microdomains are generally
structurally/functionally distinct regions of the
cell membrane, such as lipid rafts
Our example:
Flotillin is
enriched in the
DRM fraction,
just like
caveolin:
Is it a
component of
caveolae?
Is flotillin found in caveolae?
Colocalisation/interaction studies of putative
raft-associated proteins in lymphocytes:
Confocal analysis of src-kinase and Thy-1
(GPI-anchored protein)
Can we show that Nogo-66-Rec is
a raft-associated protein?
Can we show that Nogo-66-Rec is
a raft-associated protein?
There appear to be different kinds
of lipid rafts besides caveolae
Single-particle trajectories:
Dye tracking
Fluorescent Proteins
GFP - Green Fluorescent Protein
 GFP is from the chemiluminescent jellyfish Aequorea
victoria
 excitation maxima at 395 and 470 nm (quantum efficiency
is 0.8) Peak emission at 509 nm
 contains a p-hydroxybenzylidene-imidazolone
chromophore generated by oxidation of the Ser-Tyr-Gly at
positions 65-67 of the primary sequence
 Major application is as a reporter gene for assay of
promoter activity
 requires no added substrates
 now modified forms available: yellow, red, cyan and blue
fluorescent proteins
 Often used in FRET
Energy Transfer: FRET
 Effective between 10-100 Å only
 Emission and excitation spectrum must
significantly overlap
 Donor transfers non-radiatively to the
acceptor
Fluorescence Resonance
Energy Transfer
Energy Transfer
Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Absorbance
Absorbance
Wavelength
Applications of FRET
Mobility of Lipid Molecules in
the Plasma Membrane of a Cell
– “FRAP”
 FRAP – Fluorescence Recovery After
Photobleaching
 Membrane lipid (or protein) of a live cell is labeled
with a fluorescent tag.
 A spot is irreversibly bleached with a laser.
 The bleached spot is observed for recovery of
fluorescence.
- Magnitude of recovery: fraction of labeled molecules
that are mobile.
- Rate of recovery: diffusion constant.