)

Measuring chemical activity of cholesterol𝝁= 𝝁𝟎+ 𝑹𝑻𝒍𝒏𝒂

Space-filling model of b-cyclodextrin

Cyclodextrin binds lipophilic drugs, including cholesterol

Regular Solution Theory

lclc

lBll

cBcc

xxNN

xTkN

xTkNF

ln

ln

ϴ is a parameter that accounts for interactions between components (i.e., it accounts for non-ideality), μc, μl are standard chemical potentials, and xc, xl are the mole fractions of cholesterol and the phospholipid.

0.0 0.1 0.2 0.3 0.4 0.5

0.2

0.4

0.6

0.8

DOPC

0.1 0.2 0.3 0.4 0.50.00

0.25

0.50

0.75

POPC

0.20 0.25 0.30 0.35 0.40 0.45 0.500.00

0.05

0.10

0.15

0.20

0.25

SM

0.1 0.2 0.3 0.4 0.50.0

0.1

0.2

0.3

0.4

0.5

0.6

DPPC

Cholesterol Concentration, c

Cholesterol Activity, a

Θ, kT S, %

DOPC 1.17 68

POPC -2.93 64

SM -6.27 85

DPPC -1.56 79

Θ: Interaction ParameterS: Maximum Cholesterol Solubility

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.

)

Fluorescence microscopy of planar bilayer membranes

Objective

Solvent-containing Torus

Cholesterol/SM rafts form below the Tm of the SM

DOPC/DOPE (2:1), 15 mol % cholesterol and egg-SM (mostly 16:0,Tm = 41 °C)

Probe: 5 mol % rho-DOPE

Scale bar: 50 μm for A and B; 4 μm for C

Solid-gel domains are noncircular

A. 25 mol % DPPS (Tm = 55 °C) and 25 mol % cholesterolB. 25 mol % DMPE (Tm = 50 °C) and 25 mol % cholesterol

Scale bar: 4 μm

Rafts are fluid

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B. Pipette withdrawn, 5 frames after A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

Lipids are more ordered in rafts than in a

surrounding membrane

raft

Rafts dissolve at temperatures above Tm 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 tendedto accumulate at the Gibbs-Plateau border

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?

Raft formation requires saturated acyl chains and

depends on headgroupAcyl chain Domains with

cholesterol?

16:0 egg-SM Yes

18:0 SM or DSPC Yes

18:1 SM or DOPC No

18:0 DSPS No

14:0 DMPE No

16:0 and 18:0 GM1 No

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?

Rafts extend through both monolayers of a bilayer membrane

Torus

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?

Coupling of rafts in outer leaflets to lo domains of

inner leaflets

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?

Surface vs. cross-section observation of rafts

cross-section

rafts in 3D

NBD-DPPE

Rho-DOPE

GUV

raft

Composition:SM, Cholesterol, DOPC

Large raft formation as a function of cholesterol content

10% Cholesterol 40% Cholesterol

20% Cholesterol 20% SM20% SM20% SM

40% DOPC60% DOPC70% DOPC

Increasing cholesterol in a SM/DOPC GUV

0 sec

200 sec160 sec120 sec

80 sec

40 sec

Add methyl beta-cyclodextrin/cholesterol

Increasing Cholesterol

10 15 20 25 30 35 40 45 50 55

1.00

1.05

1.10

1.15

1.20

Flu

ore

sce

nce

(u

nq

ue

nch

ed

/qu

en

che

d)

T, oC

0.1% NBD-DPPE 1% Rho-DOPE

Small rafts are present at high cholesterol content in the absence of tension

Question Set # 5

Why can’t large rafts exist at high Cholesterol content?

Why don’t small rafts merge at high cholesterolcontent?

0 sec

100 sec80 sec60 sec

40 sec

20 sec

Tension promotes large rafts

20% Sphingomyelin, 40% cholesterol

Large rafts appear after liposome swelling

Swelling induces large rafts in cells at 37oC

unswollen

swollen

Cholera Toxin B – marker for GM1

OuabainCholera Toxin B

Ouabain – marker for Na/K-ATPase

in entropy

RnrN

nnkTF 22ln min

B

Raft merger is a competition between a reduction

in boundary energy and a decrease in entropy

Rafts are thicker than the surround

raft

Lateral tension increases line tension

of rafts

A

dAk

JB

JB

F

220

20 22

div2

tn

Question # 6

•Does mechanical tension increase line tension of domains that have the same thickness as the surround?

Which one is Artem?