Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes.

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.