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THREE DECADES OF QUESTIONS ON LIPID RAFTS

Whitney Stutts

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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?

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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

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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

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Video

Lipid Raftshttp://multimedia.mcb.harvard.edu/anim_innerlife.html

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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

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Raft Proteins

“True resident 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

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Why do they form?

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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

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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

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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

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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

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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

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Hijacking Viruses

HIV virus Budding may occur from lipid rafts

Influenza virus Raft-associated glycoproteins in envelope

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Disorders & Diseases

Mood disorders 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)

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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

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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

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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?

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Works Cited

Allen, John A. "Lipid raft microdomains and neurotransmitter signaling." Nature 8 (2007): 128-40.

Benarroch, Eduardo E. "Lipid rafts, protein scaffolds, and neurologic disease." Neurology 69 (2007): 1635-639.

Hamasaki, Dr. Toshikazu. "Tutorial 2, Plasma Membrane." UCLA. 22 Feb. 2009. Jacobson, Ken. "Lipid rafts: at a crossroad between cell biology and physics." Nature Cell

Biology 9 (2007): 7-13. 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, http://www.expertreviews.org/02005392h.htm

Korade, Zeljka. "Lipid rafts, cholesterol, and the brain." Neuropharmacology 55 (2008): 1265-273.

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): 597-603. Simons, Kai. "Lipid Rafts and Signal Transduction." Nature Reviews 1 (2000): 31-41. Simons, Kai. "Model Systems, Lipid Rafts, and Cell Membranes." Annu. Rev. Biophys.

Biomol. Struct. 33 (2004): 269-95. Video: Viel, A., Lue R.A., “Inner life of the cell.” The president and Fellows of Harvard

College (2007) http://multimedia.mcb.harvard.edu/anim_innerlife.html