9. )

13. Measuring chemical activity of cholesterol

14. Space-filling model of b-cyclodextrin

15. Cyclodextrin binds lipophilic drugs, including cholesterol

16. Regular Solution Theory is a parameter that accounts for interactions between components (i.e., it accounts for non-ideality), μ c, μ l are standard chemical potentials, and x c, x l are the mole fractions of cholesterol and the phospholipid.

17. DOPCPOPC SM DPPC Cholesterol Concentration, c Cholesterol Activity, a Θ, kTS, % DOPC1.1768 POPC-2.9364 SM-6.2785 DPPC-1.5679 Θ: Interaction Parameter S:Maximum Cholesterol Solubility

18. Problems Develop theories that predict strength of pair-wise interactions from experimental activity-cholesterol relationships. Generalize mean field theories, such as RST, to account for limited solubilities.

19. )

20. Fluorescence microscopy of planar bilayer membranes Objective Solvent- containing Torus

21. Cholesterol/SM rafts form below the T m of the SM DOPC/DOPE (2:1), 15 mol % cholesterol and egg-SM (mostly 16:0,T m = 41 °C) Probe: 5 mol % rho-DOPE Scale bar: 50 μm for A and B; 4 μm for C

22. Solid-gel domains are noncircular A.25 mol % DPPS (T m = 55 °C) and 25 mol % cholesterol B. 25 mol % DMPE (T m = 50 °C) and 25 mol % cholesterol Scale bar: 4 μm

23. Rafts are fluid

24. Rafts are deformable A. Pipette pulled 3-5 μm/s B. Pipette withdrawn, 5 frames after A C. Raft is circular, 9 frames after B Scale bar: 50 μm

25. Lipids are more ordered in rafts than in a surrounding membrane raft

26. Rafts dissolve at temperatures above T m of the SM Times after raising temperature: A. 1 min, B. 5 min, C. 7 min, D. 9 min, E. 23 min Membrane moved at D to show large dark domain (arrow). Large domains tended to accumulate at the Gibbs-Plateau border

27. Question set # 1 Why are rafts liquid-ordered rather than solid-ordered even though the bilayer is at a temperature below the Tm of the SM? Is the dominant effect the intercalation of cholesterol between the acyl chains that prevents the lipid tilting of the gel phase?

28. Raft formation requires saturated acyl chains and depends on headgroup Acyl chainDomains with cholesterol? 16:0 egg-SM Yes 18:0 SM or DSPCYes 18:1 SM or DOPCNo 18:0 DSPSNo 14:0 DMPENo 16:0 and 18:0 GM 1 No

29. Question Set # 2 Why does either SM or PC phase separate with cholesterol? What are the relative roles of headgroup interactions and hydrogen bonding in raft formation? Are interactions between chains the primary determinant? Does SM or PC form stoichiometric complexes with cholesterol?

30. Rafts extend through both monolayers of a bilayer membrane Torus

31. Question set # 3 Why do SM/Cholesterol domains couple between the two monolayers? Is the cause interdigitation of the saturated acyl chains of SM of one monolayer with that of the other? Or do the side chains of cholesterol in the two monolayers interact? Are there other possibilities for coupling in a pure lipid bilayer?

32. Coupling of rafts in outer leaflets to l o domains of inner leaflets

33. Question set # 4 Why are rafts small in biological cell membranes but large in lipid bilayer membranes? Do membrane proteins serve as nucleating centers for raft formation, rather than passively partition into rafts?

35. Surface vs. cross-section observation of rafts cross-section rafts in 3D NBD-DPPE Rho-DOPE GUV raft Composition: SM, Cholesterol, DOPC

36. Large raft formation as a function of cholesterol content 10% Cholesterol40% Cholesterol20% Cholesterol 20% SM 40% DOPC60% DOPC70% DOPC

37. Increasing cholesterol in a SM/DOPC GUV 0 sec 200 sec160 sec120 sec 80 sec 40 sec Add methyl beta-cyclodextrin/cholesterol

38. Increasing Cholesterol

39. 0.1% NBD-DPPE 1% Rho-DOPE Small rafts are present at high cholesterol content in the absence of tension

40. Question Set # 5 Why can’t large rafts exist at high Cholesterol content? Why don’t small rafts merge at high cholesterol content?

41. 0 sec 100 sec80 sec60 sec 40 sec 20 sec Tension promotes large rafts 20% Sphingomyelin, 40% cholesterol

42. Large rafts appear after liposome swelling

43. Swelling induces large rafts in cells at 37 o C unswollen swollen Cholera Toxin B – marker for GM1 Ouabain Cholera Toxin B Ouabain – marker for Na/K-ATPase

44. in entropy B Raft merger is a competition between a reduction in boundary energy and a decrease in entropy

45. Rafts are thicker than the surround raft

46. Lateral tension increases line tension of rafts

47. Question # 6 Does mechanical tension increase line tension of domains that have the same thickness as the surround?

48. Which one is Artem?