High-Resolution Mapping of Phase Behavior in a Ternary Lipid Mixture:  Do Lipid−Raft Phase Boundaries Depend on the Sample Preparation Procedure?

Abstract

For some time now, we have been using a fluorescence resonance energy transfer (FRET)-based strategy to conduct high-resolution studies of phase behavior in ternary lipid-raft membrane mixtures. Our FRET experiments can be carried out on ordinary, polydisperse multilamellar vesicle suspensions, so we are able to prepare our samples according to a procedure that was designed specifically to guard against artifactual phase separation. In some respects (i.e., the number and nature of two-phase regions observed), our phase diagrams are consistent with those in previously published reports. However, in other respects (i.e., overall size of miscibility gaps, phase boundary locations and their dependence on temperature), there are clear differences. Here, we present FRET data taken in dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine/cholesterol (DOPC/DPPC/Chol) mixtures at 25.0, 35.0, and 45.0 degrees C. Comparisons between our results and previously reported phase boundaries suggest that lipid-raft mixtures may be particularly susceptible to demixing effects during sample preparation.

Phase diagram of a polyunsaturated lipid mixture: Brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol

“…The precise location of boundaries was extracted from the features of the DHE → BoDIPY-PC stimulated acceptor emission data, as described [91–93]. We have prepared 9 trajectories of about 70 samples each to locate the upper Ld+Lo boundary.…”

“…5 trajectories were made to locate the boundaries for Lo+Lβ region. The number of trajectories prepared was dependent on the size of the region and we have used similar logic in selecting locations of the trajectories and data analysis as we already described in detail in ref [23, 91–93]. …”

AFM Investigations of Phase Separation in Supported Membranes of Binary Mixtures of POPC and an Eicosanyl-Based Bisphosphocholine Bolalipid

“…For example, giant unilamellar vesicles prepared from ternary lipid mixtures with co-existing liquid phases occasionally show a mixture of vesicles with and without phase coexistence by fluorescence microscopy; these results have been attributed to differences in lipid composition between vesicles 29. Similarly, a study of ternary lipid mixtures has concluded that lipid demixing leads to changes in phase diagrams when the vesicle samples are prepared by different methods 30. It has been suggested that lipid demixing artifacts may be particularly problematic for cholesterol-rich mixtures.…”

Phase behavior in a ternary lipid membrane estimated using a nonlinear response surface method and Kohonen’s self-organizing map

“…For instance, Buboltz et al investigated phase behavior in mixtures of DPPC, DOPC, and Ch using FRET measurements [17] and also encountered the problem of the number of data sets required for interpretation of their results. To address this issue, they examined 1294 kinds of lipid compositions and prepared a ternary phase diagram.…”

“…Fluorescence analyses such as Förster resonance energy transfer (FRET) and fluorescence anisotropy are also employed to identify the lipid phase behavior of membranes [14,[16][17][18][19]. Because these measurements do not require preparation of a giant unilamellar vesicle, a wider range of lipid compositions can be examined than with CFM.…”