Lipid composition dictates membrane thickness, which in turn can influence membrane protein activity. Lipid composition also determines whether a membrane demixes into coexisting liquid-crystalline phases. Previous direct measurements of demixed lipid membranes have always found a liquid-ordered phase that is thicker than the liquid-disordered phase. Here we investigated non-canonical ternary lipid mixtures designed to produce bilayers with thicker disordered phases than ordered phases. The membranes were comprised of short, saturated (ordered) lipids; long, unsaturated (disordered) lipids; and cholesterol. We found that few of these systems yield coexisting liquid phases above 10 °C. For membranes that do demix into two liquid phases, we measured the thickness mismatch between the phases by atomic force microscopy and found that not one of the systems yields thicker disordered than ordered phases under standard experimental conditions. We found no monotonic relationship between demixing temperatures of these ternary systems and either estimated thickness mismatches between the liquid phases or the physical parameters of single-component membranes comprised of the individual lipids. These results highlight the robustness of a membrane’s liquid-ordered phase to be thicker than the liquid-disordered phase, regardless of the membrane’s lipid composition.

Micron-scale coexisting Lo and Ld liquid phases can appear in lipid bilayers composed of a ternary mixture of a low-melting temperature lipid, a high-melting temperature lipid, and cholesterol. A priori, temperatures at which membranes demix, Tmix, are not simply related to differences in thicknesses, Δh, between Lo and Ld phases. Here, we use fluorescence microscopy to measure Tmix and we use atomic force microscopy at 22°C to measure Δh for a series of bilayers composed of different ratios of the three components. Our data illustrate cases in which a change in Tmix or Δh does not result in a change in the other parameter. The data provide a context in which to evaluate recent reports of a correlation between Tmix and Δh.

The composition of single MCF-7 breast cancer cells is characterized using 2-D CE. Individual MCF-7 cells were aspirated into a 30 mum inner diameter fused-silica capillary and lysed by contact with an SDS-containing buffer. Proteins and other primary amines were fluorescently labeled on-column using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled components were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by MEKC. Analytes were detected in a sheath-flow cuvette using LIF. The expression profiles for MCF-7 cellular homogenate and a single MCF-7 cell are compared. As a proof-of-principle investigation, variation in expression was also compared within and between G1 and G2/M cell cycle phases for MCF-7 cells. Following their treatment with the viable nuclear stain Hoechst 33342, MCF-7 cells were sorted by flow cytometry on the basis of their ploidy. Sorted cells were then analyzed by 2-D CE. The degree of variability was >2.5 times larger between cells of different phases than between cells of the same phase. In typical 1 h 2-D CE separations using MCF-7 cells, over 100 components are resolved.

This influx of information provides new opportunities for understanding the chemistry of these proteins. Using a structural bioinformatics approach, we have determined a strong asymmetry in the charge distribution of these proteins. For the outward-facing amino acids of the beta barrel within regions of similar amino acid density for both membrane leaflets, the external side of the membrane contains more than three times the number of charged amino acids as the internal side of the membrane. Moreover, the lipid bilayer of the outer membrane is asymmetric, and the overall preference for amino acid types to be in the external leaflet of the membrane correlates roughly with the hydrophobicity of the membrane lipids. This preference is demonstrably related to the difference in lipid composition of the external and internal leaflets of the membrane. The charge asymmetry of proteins in the outer membrane has important implications for how we understand the mechanism of outer membrane protein insertion.

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