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

TL;DR

This study uses FRET to map phase behavior in ternary lipid mixtures, revealing that sample preparation procedures can influence observed phase boundaries and demixing effects.

Contribution

It demonstrates that lipid-raft phase boundaries depend on sample prep, challenging previous assumptions of their invariance.

Findings

Phase diagrams are consistent with prior reports in some aspects.

Overall size of miscibility gaps varies with sample prep.

Lipid-raft mixtures are sensitive to demixing during preparation.

Abstract

For some time now, we have been using a FRET-based strategy to make 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 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 DOPC/DPPC/Cholesterol mixtures at 25.0, 35.0 and 45.0oC. Comparisons between our results and previously reported phase boundaries suggest that lipid-raft mixtures may be particularly susceptible to demixing effects during sample preparation.