Fluorescence Resonance Energy Transfer between Lipid Probes Detects Nanoscopic Heterogeneity in the Plasma Membrane of Live Cells
Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C^sub 18^ is greater with long-chain, saturated acceptor probes such as DiI-C^sub 16^ than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C^sub 16^ and DiI-C^sub 16^ is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C^sub 16^. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.
The existence of lateral inhomogeneities or domains in the lipid portion of the plasma membrane is an issue of substantial interest and controversy . Studies on detergent-resistant membranes led to the hypothesis that lateral segregation of liquid ordered (Lo) and liquid disordered (Ld) lipids in the plasma membrane plays an important role in signal transduction, protein sorting, and membrane transport . These cholesterol-dependent ordered membrane domains that selectively contain lipids and proteins are commonly called lipid rafts. However, the difficulty in observing lipid membrane domains at optical resolution has made it challenging to relate biochemical evidence for lipid heterogeneity, such as that from detergent-dependent fractionation, to properties in live cells. Detecting segregated membrane domains in live cells usually requires large-scale cross-linking of lipid raft components and/or low temperature (7-9). Some advanced methods with high spatial (nanometer) and temporal (millisecond) resolution, such as single particle tracking and video microscopy, nanosecond depolarization homo-FRET (13), and high-resolution electron microscopy , have provided evidence for membrane domains in intact plasma membranes. However, direct evidence for the existence of lipid rafts in live cells is largely based on measurements of clustering or diffusion of putative raft proteins rather than on measurements of the lipids themselves. A recent exception is the detection of the ordered and disordered lipid phases in live cells with ESR measurements of spin-labeled lipid probes (16).The ordered state of lipid rafts is based on preferential interactions between cholesterol and phospholipids with long saturated acyl chains such as sphingolipids. Correspondingly, lipid probes with different acyl/alkyl chains are expected to partition differently between ordered domains and disordered regions of the bilayer. In our study, we investigated the lateral distributions of fluorescent lipid probes in the outer leaflet of live cell membranes using FRET between carbocyanine derivatives with differing alkyl chains. Carbocyanines label the outer leaflet of cell membrane with negligible transbilayer flip-flop (17), making them ideal outer leaflet probes. Furthermore, their high photostability, large extinction coefficients, and partition coefficients that strongly favor lipid over aqueous environments (K^sub p^ ~ 10^sup 3-4^ for DiO-C^sub 6^) (18) make them highly suitable for fluorescence measurements in live cell membranes. FRET is particularly useful for monitoring inhomogeneities in the lateral organization of lipid bilayers on a spatial scale intermediate between the ~300-nm scale of light microscopy and the nearest neighbor (0.1 nm) scale of Stokes quenching of fluorescent or brominated lipids by spin labels (19,20). Previous measurements of FRET between lipidanchored fluorescent proteins at the inner leaflet of plasma membranes provided evidence for inhomogeneity on the scale of tens of nanometers (21), illustrating the value of this noninvasive technique for studying membrane domains with dimensions below optical resolution.
Our measurements of FRET between carbocyanine lipid probes in the plasma membrane of live cells provide strong evidence for lateral lipid inhomogeneities that are sensitive to perturbations that enhance or reduce Lo/Ld segregation. These results correlate well with lipid partitioning as measured by detergent resistance. The method we describe provides a novel approach to studying membrane lipid heterogeneity in live cells that is relevant to cell functions.