40s 50s. 70s. Membrane Rafts

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1 40s 50s 70s Membrane Rafts

2 Membrane Microdomains Raft is a specific type of microdomain sphingolipid/cholesterol rich region Separation of discrete liquid-ordered and liquid-disordered phase domains occurring with sufficient amounts of cholesterol Microdomain formation is believed to be involved in following cellular processes: Cell sorting Signal transduction Endocytosis Calcium homeostasis And others Rafts: liquid ordered domain lipids are fluid in that they have a high degree of lateral diffusion, but the acyl chains are closed packed and ordered. Glycosphingolipids (particularly sphingolmyelin and glycosylphosphoinositol- GPI anchored proteins preferentially partition into rafts.)

3 The debate: Rafts in model membranes vs Rafts in Biological Membranes Origin: TritonX 100 insoluble components isolated from biological membranes: Detergent Resistant Membranes DRM. Does DRM always equal a raft TritonX100 can solubilize DOPC:chol but Not DPPC:chol

4 Sphingomyelin cholesterol interactions Sphingomyelin POPC

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6 December reviews on domain formation in model membranes and physical properties that underlie raft formation 2 reviews to describe techniques used for studying rafts (FRET) and uncertainty for detecting rafts in cell membranes Raft Function in Cells: 4 on signal transduction(ige receptor signaling, Growth factors, Ras signaling) Ceremide Raft function in apoptotic signaling 3 reviews on raft involvement in Endocytosis (mammalian viruses, bacterial infections, bacterial toxins) 2 reviews of caveloae

7 Membrane raft Organization DRMs detergent resistant membranes DIGs detergent insoluble glycolipidenriched membranes GEMs glycolipid enriched membranes TIFFs Triton insoluble membranes Raft is more generic as the microdomain can be caused by protein association, not just physical properties of the lipids themselves Rafts may or may not contain caveolin Caveolin1, caveolin2, caveolin 3, hemagglutinin and GPI anchored proteins serve as markers for raft formation

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9 A: liquid domains enriched in cholesterol and sphingolipids large scale > 50 nm B: lipid shells, small dynamic, regulated processes C: mosaic of domains, maybe regulated by cholesterol-based mechanism D: small dynamic multimeric lipid assemblies, dynamic and transient

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12 Protein sorting Melittin: 26 aa cationic bee venom: channels Role of structural and mechanical properties of bilayers on peptidelipid partitioning

13 1:1:1 mixture of DOPC:SPM:CHOL, the detergent insoluble fraction has a thickness that is 9Å greater than that of the DSM Role of Bilayer thickness in protein-lipid interactions: possible role in sorting of proteins via hydrophobic mismatch of the transmembrane domain (TMD). Hydrophobic mismatch: if there is a mismatch between the length of the TMD and the hydrocarbon thickness, then the bilayer would need to deform to prevent exposure of the hydrophobic amino acids to water. This would be energetically unfavorable. So, if the protein can move to a raft of different thickness, there would be a driving force for such partitioning.

14 Bio significance: in GOLGI, proteins with short TMDs reside in non raft regions, whereas proteins with longer TMDs reside in raft regions destined to the plasma membrane (rich in cholesterol and SPM). Length of TMD has been indicated to be an important factor in controlling protein trafficing.

15 Experimental studies of peptide sorting by length Thermal kt = 0.6 kcal/mol at 37 o C.

16 Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are they static large structures? Are they small transient structures? FRET and FRET based Microscopy Techniques

17 FRET fluorescence resonance energy transfer

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