Plasmon-waveguide Resonance Studies of Lateral Segregation of Lipids and Proteins into Microdomains (Rafts) in Solid-supported Bilayers

Abstract

Plasmon-waveguide resonance (PWR) spectroscopy has been used to examine solid-supported lipid bilayers consisting of dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), sphingomyelin (SM), and phosphatidylcholine/SM binary mixtures. Spectral simulation of the resonance curves demonstrated an increase in bilayer thickness, long-range order, and molecular packing density in going from DOPC to POPC to SM single component bilayers, as expected based on the decreasing level of unsaturation in the fatty acyl chains. DOPC/SM and POPC/SM binary mixtures yielded PWR spectra that can be ascribed to a superposition of two resonances corresponding to microdomains (rafts) consisting of phosphatidylcholine-and SM-rich phases coexisting within a single bilayer. These were formed spontaneously over time as a consequence of lateral phase separation. Each microdomain contained a small proportion (<20%) of the other lipid component, which increased their kinetic and thermodynamic stability. Incorporation of a glycosylphosphatidylinositol-linked protein (placental alkaline phosphatase) occurred within each of the single component bilayers, although the insertion was less efficient into the DOPC bilayer. Incorporation of placental alkaline phosphatase into a DOPC/SM binary bilayer occurred with preferential insertion into the SM-rich phase, although the protein incorporated into both phases at higher concentrations. These results demonstrate the utility of PWR spectroscopy to provide insights into raft formation and protein sorting in model lipid membranes.The classical textbook model of biomembrane structure, usually referred to as the fluid-mosaic model, envisions a twodimensional solution of integral membrane proteins in a homogeneous lipid solvent, albeit one composed of many molecular lipid species and possessing inside-outside asymmetry with respect to both protein and lipid components. However, in recent years, there has been a growing recognition that lateral segregation of lipids and proteins occurs within regions of biomembranes called rafts (cf. Refs. 1 and 2). Along with caveolae, which are invaginations of raft regions stabilized by interactions with oligomers of the protein caveolin, these microdomains have been suggested to play important roles in cell polarity, protein sorting, signal transduction, and membrane trafficking (cf. Refs. 3 and 4).One of the key properties of rafts is their high content of sphingomyelin (SM) 1 and cholesterol, which leads to their being organized into what are referred to as liquid-ordered domains. These are characterized by being more highly ordered and somewhat thicker than the surrounding liquid-disordered regions of the membrane. This is a consequence of the ordering influence of cholesterol and the presence in SM of a larger proportion of saturated fatty acyl chains. However, it should be noted that studies of model membranes have shown that microdomain formation also occurs in phosphatidylcholine (PC)/SM mixtures in the absence of cholesterol (2). These li...

Ligand Modulation of Lateral Segregation of a G-Protein-Coupled Receptor into Lipid Microdomains in Sphingomyelin/Phosphatidylcholine Solid-Supported Bilayers

“…This heterogeneity varies from experiment to experiment. However, as we have shown previously (24), this does not affect the properties of the individual microdomains obtained by the deconvolution process.…”

“…Thus, the latter component is thicker and more densely packed than the former. These values can be compared with a thickness of 5.4 nm and a surface area per molecule of 0.48 nm 2 for a pure POPC bilayer, and a thickness of 6.1 nm and a surface area per molecule of 0.39 nm 2 for a pure SM bilayer (24), clearly indicating that the lower angle component can be attributed to the POPC-rich phase and the higher angle component to the SM-rich phase, as previously concluded.…”

“…We have also used PWR spectroscopy to examine solid-supported lipid bilayers consisting of pure lipids (DOPC, POPC and SM), and PC:SM binary mixtures (24). We have shown that a single lipid bilayer formed from the PC:SM mixture consists of microdomains enriched in the SM component, coexisting with PC-rich domains, whose different optical properties allow observation of separate resonances in a PWR experiment.…”

Use of plasmon waveguide resonance (PWR) spectroscopy for examining binding, signaling and lipid domain partitioning of membrane proteins

“…We also have investigated the effects of membrane microdomains (lipid rafts) on GPCR function again using the hDOR as the example Salamon et al 2005). In these experiments we first demonstrated that if we utilized a 1:1 mixture of dioleoylphosphatidylcholine (DOPC) and sphingomyelin (SM) we could unequivocally demonstrate using PWR spectroscopy that the lipid bilayer spontaneously segregated into two separate domains, the more fluid DOPC rich domain (5.2 nm thickness), and the more rigid sphingomyelin rich domain (5.9 nm thickness) .…”

4 Electrochemistry of Biomimetic Membranes

“…The PWR spectroscopy has also been used to monitor the formation of microdomains in mixed sphingomyelin-DOPC mixtures (called lipid rafts) and the sorting of receptors into each microdomain. 153 The microdomain size evaluated from the lateral resolution of the PWR sensor is very high (100-300 m). While providing useful structural information on (lipid bilayer) -(integral protein) -(peripheral protein) systems, this technique cannot provide direct evidence for the functional activity of these systems and a verification that the differential capacitance of the lipid film is that expected for a well-behaved lipid bilayer.…”