1. Three decades of Questions on Lipid Rafts
Whitney Stutts

2. Outline What are lipid rafts? Why do they form?
What methods are used to study lipid rafts?
What effects do they have on eukaryotes?
Why all the controversy?

3. What are lipid rafts?
“Lipid rafts are small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes.”
~ 2006 Keystone Symposium

4. What are lipid rafts?
Cholesterol and sphingolipid-enriched membrane microdomains or platforms
Cholesterol levels double
Sphingomyelin levels elevated by 50%
Concentrate and segregate proteins within the plane of the bilayer
More ordered and tightly packed than surrounding bilayer
Float freely in Lc bilayer

5. Video
Lipid Rafts ml

6. Two types of lipid rafts
Caveolae: small, flask- shaped invaginations of the plasma membrane enriched in caveolin
Planar lipid rafts: found in neurons and enriched in flotillin
Caleolin and flotillin recruit signaling proteins
Signaling can be promoted or dampened

7. Raft Proteins “True resident proteins” Signaling proteins
GPI-anchored proteins-prion protein (PrPc)
Caveolin
Flotillin
Signaling proteins
G-protein, non-receptor tyrosine kinases
Cytoskeletal/Adhesion proteins
actin, myosin, vinculin, cofilin, cadherin, ezrin

8. Why do they form?

9. Why do they form? Cholesterol
Cholesterol is the dynamic “glue” that holds the raft together
Saturation
Hydroxyl H-bonding with amide
Up to 25% of cholesterol is found in the brain…CNS?
When removed, most proteins dissociate from rafts

10. Misconception
Rafts contain only phospholipids with fully saturated acyl chains….FALSE!
Most glycerophospholipids in membrane rafts contain at least one monounsaturated acyl chain
Sphygomyelin- usually saturated chains but when unsaturated the DB is located at C15

11. Why do rafts form?
Driving force- line tension: energy required to create a boundary between the raft and the surrounding membrane
Raft thickness Hydrophobic mismatch
contrast in thickness line tension
Results in larger, more circular rafts which reduce line tension and energetic cost of the raft interface length

12. What methods are used to study lipid rafts?
DRM Isolation - lipid rafts are insoluble in cold non-ionic detergents (Triton X-100)
Electon microscopy - determines location of raft components and can detect clustering of proteins
FRET – used to determine whether two raft components are spatially close
FRAP - probe for the association of proteins to lipid rafts and study diffusion of proteins in lipid rafts
Manipulation of cholesterol
Sequestration
Depletion or removal
Inhibition of cholesterol synthesis

13. What effects do rafts have on eukaryotes?
Organizing centers-assembly of signaling molecules
Signaling can be promoted or dampened
Effects membrane fluidity
Acyl chain fluidity
Lateral diffusion
Involved in trafficking of membrane proteins
Regulation of neurotransmission and receptor trafficking
Neurotrophin receptors embedded in rafts

14. Hijacking Viruses HIV virus Influenza virus
Budding may occur from lipid rafts
Influenza virus
Raft-associated glycoproteins in envelope

15. Disorders & Diseases Mood disorders Alzheimer’s disease Prion disorder
Therapeutic efficacy of antidepressants
Alzheimer’s disease
Platforms for production of amyloid-β (neurotoxic protein)
Prion disorder
Normal prion protein (PrPc) is converted to abnormal proteins (PrPsc) in lipid rafts
(GPI anchor required)

16. Why all the controversy?
Problems with biomembranes
Lipid rafts are too small to be resolved by light microscopy
Difficult to study lipid rafts in intact cells
Not in thermodynamic equilibrium

17. Why all the controversy?
Problems with synthetic membranes
Lower concentration of proteins
Difficult to model membrane-cytoskeletal interactions
Lack natural lipid asymmetry
Studied under equilibrium conditions

18. More Questions What are the effects of membrane protein levels?
What is the physiological function of lipid rafts?
What effect does flux of membrane lipids have on raft formation?
What effect do diet and drugs have on lipid rafts?
What effect do proteins located at raft boundaries have on lipid rafts?

19. Works Cited
Allen, John A. "Lipid raft microdomains and neurotransmitter signaling." Nature 8 (2007):
Benarroch, Eduardo E. "Lipid rafts, protein scaffolds, and neurologic disease." Neurology 69 (2007):
Hamasaki, Dr. Toshikazu. "Tutorial 2, Plasma Membrane." UCLA. 22 Feb
Jacobson, Ken. "Lipid rafts: at a crossroad between cell biology and physics." Nature Cell Biology 9 (2007):
Jacques Fantini, Nicolas Garmy, Radhia Mahfoud and Nouara Yahi (2002) Lipid rafts: structure, function and role in HIV, Alzheimer’s and prion diseases. Exp. Rev. Mol. Med. 20 December,
Korade, Zeljka. "Lipid rafts, cholesterol, and the brain." Neuropharmacology 55 (2008):
Luckey, Mary. Membrane Structural Biology : With Biochemical and Biophysical Foundations. New York: Cambridge UP, 2008.
Pike, Linda J. "The Challenge of Lipid Rafts." Journal of Lipid Research Oct (2008): 1-17.
Simons, Kai, and Ehehalt, R. "Cholesterol, lipid rafts, and disease." The Journal of Clinical Invesigation 110 (2002):
Simons, Kai. "Lipid Rafts and Signal Transduction." Nature Reviews 1 (2000):
Simons, Kai. "Model Systems, Lipid Rafts, and Cell Membranes." Annu. Rev. Biophys. Biomol. Struct. 33 (2004):
Video: Viel, A., Lue R.A., “Inner life of the cell.” The president and Fellows of Harvard College (2007)