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. 2008 Jan 8;105(1):124-8.
doi: 10.1073/pnas.0702970105. Epub 2008 Jan 2.

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

Marcus D Collins  1 , Sarah L Keller
Affiliations

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

Marcus D Collins et al. Proc Natl Acad Sci U S A. .

Abstract

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal-Mueller planar bilayers in which one leaflet's composition would phase-separate in a symmetric bilayer and the other's would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Phase behavior of symmetric bilayers in both giant unilamellar vesicles and planar bilayers. (a) Phase boundary of liquid–liquid immiscibility region for vesicles of the ternary lipid mixture DiPhyPC, DPPC, and Chol at 25°C (15). Following the arrow from left to right, vesicles display one uniform phase, then dark (Lo) domains on a bright (Ld) background, and then bright domains on a dark background. (b–d) Fluorescence micrographs of symmetric planar bilayers ≈180 μm in diameter at 24 ± 0.5°C follow the same phase behavior described above. Domains in one leaflet are in registration with domains in the opposite leaflet, as in symmetric vesicles. The compositions of lipids are as follows: composition A, 61.3/5.3/33.3 DiPhyPC/DPPC/Chol (b); composition B, 33.3/33.3/33.3 DiPhyPC/DPPC/Chol (d); and composition C, 53.3/13.3/33.3 DiPhyPC/DPPC/Chol (c).
Fig. 2.
Fig. 2.
Asymmetric planar membranes ≈180 μm in diameter, with leaflets that mimic a cell membrane inner leaflet or that have composition A, B, or C from Fig. 1a. An asymmetric A/B membrane, with composition A in leaflet 1 and B in leaflet 2, contains liquid-ordered domains in both leaflets. Three distinct fluorescence intensities appear because three distinct bilayer-spanning phases are present. Domains also appear in asymmetric inner/B membranes but not in asymmetric A/C or inner/C membranes.
Fig. 3.
Fig. 3.
Free-energy and composition models of asymmetric bilayers. (a) Free energy vs. composition for a lipid membrane containing two components with a positive interaction energy. Compositions that lie between the two minima separate into coexisting Lo and Ld phases. (b and c) Sketches of asymmetric A′/B′ membranes in which both leaflets have phase-separated. (a Inset) Interactions between leaflets transforms the simplest sketch such that compositions in each leaflet are affected by the opposite leaflet. (b) Each leaflet contains three distinct lipid compositions such that the membrane as a whole contains six compositions. (c) An equivalent picture is of three distinct membrane phases of asymmetric compositions. (d) Contour plot of free energy with respect to φ1 and φ2, based on a simple Landau free energy with coupled compositions (17). A point with an initial composition at the white circle can phase-separate into three different compositions, a slightly asymmetric bilayer in the Ld phase (Ldb), a slightly asymmetric bilayer in the Lo phase (Lob), and a highly asymmetric bilayer (mixed). In contrast, a point with an initial composition at the white square does not lower its free energy and does not phase-separate.
Fig. 4.
Fig. 4.
Asymmetric planar membranes with dye in only one leaflet exhibit three distinct fluorescence levels. All membranes have one leaflet of composition near point A in Fig. 1. Specifically, the molar ratio of DiPhyPC/DPPC/Chol in the first leaflet is 56.3/10.5/33.3 in parts a and b, 57.9/8.8/33.3 in c, and 60.0/6.7/33.3 in d. In all cases, the composition of the second leaflet is at point B in Fig. 1 (33.3/33.3/33.3). The first leaflet contains 0.1% of the dye Texas red DPPE in a, c, and d. The second leaflet contains the dye in b.
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