Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Abstract

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.

FIGURE 1

Fluorescence micrographs of planar-supported bilayers mimicking the inner leaflet lipid composition of the mammalian PM. Bilayers were composed of equimolar amounts of porcine brain PC and PE (A) or PC, PE, and PS (B) and cholesterol as indicated. Bilayers were formed by the LB/LS method on quartz slides at room temperature and stained with 0.5% Rh-DPPE in the LS monolayer only. The white bar represents 10 μm.

FIGURE 2

Asymmetric planar-supported bilayer systems with coexisting liquid phases in the proximal layer. Bilayers are supported on DPS polymers that are linked to quartz supports. The proximal monolayer contains stable l_o phase domains in an l_d phase lipid background. The transbilayer effect of the proximal domains is monitored by examining the distribution and diffusion of dyes in the distal layer. The presence of these domains may induce domains in the distal monolayer (A) or may have no effect on the distal monolayer (D). Bilayers were made by the LB/VF technique at room temperature. The LB monolayer was composed of bPC/bSM/cholesterol (2:2:1) + 3% DPS. The vesicles contained either bPC, bPE, bPS (1:1:1) + 20% cholesterol (B and C), or POPC + 20% cholesterol (E and F). 0.02% Rh-DPPE was added to the LB monolayer only (B and E) or 0.5% NBD-DMPE was added to the vesicles only (C and F). The same regions of double-labeled bilayers are shown in B and C, and E and F, respectively. The white bars represent 20 μm.

FIGURE 3

Formation of planar-supported bilayers containing asymmetric l_o phase domains. Bilayers were made by the LB/VF technique at room temperature. The LB monolayer was composed of bPC/bSM/cholesterol (2:2:1) + 3% DPS (A and B) or without DPS (C). The vesicles contained either POPC + 20% cholesterol (A), bPC, bPE, bPS, and cholesterol (B), or POPC only (C). Formation of the bilayer was monitored by TIRFM with 0.5% Rh-DPPE added to the vesicles only (panels on the left). The stability of the LB monolayer during incubation with vesicles was examined by imaging with 0.5% Rh-DPPE added to the LB monolayer only (micrographs on the right). The white bar represents 20 μm.

FIGURE 4

Effects of lipid phase and DPS polymers on the stability of LB monolayers during incubation with vesicles to form asymmetric planar-supported bilayers. Bilayers were made by the LB/VF technique at room temperature. The LB monolayer was stained with 0.5% Rh-DPPE and composed of POPC (A), POPC + 3% DPS (B), DPPC/cholesterol (C), or DPPC/cholesterol + 3% DPS (D). The vesicles contained only POPC in all cases. The white bar represents 20 μm.

FIGURE 5

Degradation of lipid asymmetry in planar-supported bilayers monitored by FLIC microscopy. Bilayers were made by the LB/VF technique on 4-oxide FLIC chips at room temperature and stained with 0.5% Rh-DPPE in the vesicles only. (A) Fluorescence micrograph of an asymmetrically stained POPC bilayer on a 4-oxide FLIC chip. Each square on the chip is 5 × 5 μm². (B) Histogram of measured average distances of dyes from the proximal face of the supported POPC bilayer immediately after its completion. The fraction f_D of dye remaining in the distal layer is high at this time. A completely randomized bilayer would have an average dye distance 〈d〉 of 2 nm as indicated by the dashed line. (C) Time course of fraction of Rh-DPPE remaining in the distal monolayer of an asymmetrically stained supported POPC bilayer.

FIGURE 6

Single molecule tracking in asymmetric planar-supported bilayers. Bilayers were made by the LB/VF technique at room temperature. The LB monolayer was composed of bPC/bSM/cholesterol (2:2:1) + 3% DPS, and the vesicles were composed of POPC/cholesterol (4:1). In A, the vesicles were stained with 0.01% A647-DMPE to examine the overall distribution of the dye in the distal monolayer. In B, only 10⁻⁵% A647-DMPE was added to the vesicles for resolving and measuring the diffusion of single molecules. In C, 0.01% Rh-DPPE was added to the LB monolayer to confirm the position of l_o phase domains in the proximal monolayer. B and C show the same region of a bilayer viewed through the A647-DMPE and Rh-DPPE channels, respectively. (D) Expanded view of boxed area in C. Trajectories from diffusing A647-DMPE molecules are overlaid onto the image of the proximal monolayer to distinguish between lipids diffusing in opposite different phases. (E) Twelvefold magnified view of the two trajectories shown in (D). (F) Two spatial fluorescence intensity distributions originating from one position each of the traces in D and E. Pixel size, 0.25 μm. Scale bars are 10 μm in A–C, 2 μm in D, and 0.25 μm in E.

FIGURE 7

Fits of SPT data using MSD (left) and CDF (right) methods. Bilayers were made by the LB/VF technique at room temperature. The LB monolayer was composed of bPC/bSM/cholesterol (2:2:1) + 3% DPS, and the vesicles were composed of POPC/cholesterol (4:1). Four sets of trajectories were analyzed: molecules diffusing in the distal monolayer opposite the l_d phase of the proximal monolayer (A), molecules diffusing in the distal monolayer opposite l_o phase domains in the proximal monolayer (B), molecules diffusing in the proximal monolayer within the l_d phase (C), and molecules diffusing in the proximal monolayer within l_o phase domains (D). The panels on the left show the MSD (Eq. 5) of the pooled trajectories up to a time lag of 150 ms. A weighted least-squares fit (solid line) was applied to the first four steps of the MSD to give D_msd (Eq. 6). The panels on the right show the same data sets evaluated using the CDF of the square displacements (Eq. 7) with a time lag of 32 ms. Small dots show 1-P of the experimental data. Curves are fits to the CDF with α fixed at 1 (single diffusion coefficient D₀, solid line) or the faster fraction α as a free variable (two diffusion coefficients D₁ and D₂, dashed line). The dotted lines are fits to the anomalous diffusion model (Eq. 9). Results from the first three fitting techniques are reported in Table 1.

FIGURE 8

Diffusion coefficients of NBD-DMPE and A647-DMPE in the proximal and distal layers of single-phase symmetric bilayers of different fluidity (first three data groups) and two-phase asymmetric bilayers composed of indicated lipids in the proximal (lower labels) and distal (upper labels) layers (last two data groups). All proximal monolayers contain 3 mol % DPS. The single molecule diffusion coefficients shown are derived from fits to the MSD and to the CDF for normal diffusion of one fraction.