Figure 1.
Algorithm scheme and influence of algorithm parameters.
(A) Flow chart illustrating the 2-step minimization procedure applied to simulate the lattice dynamics and to calculate the ordering states of the lipids. (B) Box-plot depicting the influence of the control parameters NJ (counting the number of updates of the ordering state carried out per lipid to minimize the ordering energy J) and NW (counting the number of pairwise lipid switches per lattice site in the minimization of the interaction energy W) on the phase separation of the model membrane. The parameter NJ adopted different values between 10 and 1000 represented by the different boxes. For each value of NJ the parameter NW adopted the values 1, 5, 10, 20, 50 and 100. For all combinations of NJ and NW the percentage of the Ld phase (= extractable membrane fraction) was computed. The edges of the boxes indicate the lower and upper quartile, the horizontal bar inside indicates the median and the whiskers indicate the full range of the calculated values at fixed NJ and NW varied.
Figure 2.
Lipid mobility and phase diagram.
Simulation of lipid diffusion and lipid phases in GUV’s with varying lipid composition. (A) Simulated (filled markers) and measured (open markers) average diffusion coefficients in monolayers with 25 different mixtures of CH, PC and SM. Triangles facing up mark values of a liquid-disordered phase, triangles facing down of a liquid-ordered phase. Mixtures with both values show biphasic behavior, mixtures with either of both show respective monophasic behavior. The percentage of each lipid in the mixture is indicated at the bottom (CH—PC—SM).
(B) Ternary phase diagram for mixtures of CH, PC and SM. The bold lines mark regions of lipid compositions for which the model predicts monophasic and biphasic behavior. In the monophasic Lo or Ld compositions more than 90% of the lipids are resident in the respective ordering state, whereas in the biphasic mixtures more than 10% of the lipids are resident in both ordering states thus favoring the formation of segregated microdomains. The triangles mark the measured lipid mixtures shown in (A) and indicate their measured phase state (up-facing triangle: Ld, down-facing triangle: Lo, star: biphasic) which not always matches our model prediction.
Figure 3.
Membrane configurations and lipid extraction.
Representative domain structures (A—C), EMF, LSR, and lipid composition (D—F) of BS-extractable membrane patches for three different life-times (=simulations times) of τ = 0.1, 1 and 10 ms. (A—C): At short life-times ((A), τ = 0.1 ms) maze-like structures are formed comprising closed Lo nanodomains (bright colors) enriched in CH (blue) and SM (red) which are imbedded into a sea of interconnected Ld nanodomains (dark colors) enriched in PC (green). With increasing life-times (B, C), the nanodomains merge with each other under formation of larger domains. (D—F) Shown as a function of the patch size are EMF (dashed curves), LSR (full curves) and bile composition (area in the background). While the absolute LSR varies, the lipid composition of BS extractable patches remains remarkably stable at varying patch sizes and fits well with observed lipid composition of the normal bile.
Table 1.
Lipid composition of the bile.
Figure 4.
Influence of membrane lipid composition on lipid extraction.
Simulated lipid extraction at varying CH and PC content of the exoplasmic leaflet of the apical membrane. Simulations refer to a life-time of 1 ms. Shown are the EMF (black curves) and the LSR (grey curves, logarithmic scale) as a function of CH content (A) or PC content (B) in the membrane for the extraction of single lipids and patches of 19, 169 and 631 lipids. The mean lipid composition of the soluble Ld nanodomains is depicted as blue (CH), green (PC) and red (SM) areas. (A) The CH content of the outer leaflet was varied from 0–70% at constant PC:SM ratio of 3:1. (B) The PC content of the outer leaflet was varied from 0–100% at a CH:SM ratio of 7:3.
Figure 5.
Illustration of the molecular mechanisms proposed for biliary lipid secretion.
Lipids arrive at the apical membrane of the hepatocyte by various modes of transportation (vesicular, lipid-exchange proteins, lateral membrane transport). Bile salts (BS) are secreted into the canaliculus by the bile-salt export pump (BSEP). Inter-leaflet lipid exchange (indicated by the black double-arrows) by various ABC transporters and P-type ATPases (e.g. MDR1, MDR3, MRP1) establish an asymmetric lipid distribution between the inner and outer hemi-leaflet with CH, PC and SM enriched in the exoplasmic leaflet in a mixture which allows the spontaneous formation of Lo and Ld nanodomains. BS preferentially solubilize lipids of the Ld nanodomains. Mechanisms of lipid extraction currently discussed in the literature envisage (A) extraction of single membrane lipids (mostly PC) by BS micelles under formation of mixed micelles or (B) exo-vesiculation of membrane patches which form mixed micelles. Primary micelles may rapidly fuse to larger lipid vesicles (micelle-to-vesicle transition). The results of our simulation strongly favor the secretion mechanism B (lipid-patch extraction).