The Plasmamembrane and Lipid Rafts 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology UCT Medical School Room Phone:

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 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?

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 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 Å only  Emission and excitation spectrum must significantly overlap  Donor transfers non-radiatively to the acceptor

Fluorescence Resonance Energy Transfer Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 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.