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Three decades of Questions on Lipid Rafts

Three decades of Questions on Lipid Rafts. Whitney Stutts. 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?. What are lipid rafts?.

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Three decades of Questions on Lipid Rafts

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  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 Lcbilayer

  5. Video • Lipid Rafts http://multimedia.mcb.harvard.edu/anim_innerlife.html

  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” • 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 • Budding may occur from lipid rafts • Influenza virus • Raft-associated glycoproteins in envelope

  15. 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)

  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): 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, RadhiaMahfoud and NouaraYahi (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

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