The Plasmamembrane

and Lipid Rafts

08/2007

Lecture by Dr. Dirk Lang

Dept. of Human BiologyUCT Medical School

Room 6.10.1Phone: 406-6419E-Mail: DIRK.LANG@UCT.AC.ZA

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:

1) Vesicle budding and protein sorting?

2) Vesicle transport and interaction with cytoskeleton?

3) Membrane fusion and exocytosis?

How to analyse lipid rafts?

1) Fractionation of cells and isolation of detergent-resistant membrane (DRM) fraction

2) Non-disruptive microscopy methods:Single particle trackingColocalisation 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 onlyEmission and excitation spectrum must

significantly overlapDonor transfers non-radiatively to the

acceptor

Fluorescence Resonance Energy

TransferEnergy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

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.