Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes

• Published in: Biochimica et biophysica acta Vol. 1818; no. 7; pp. 1777 - 1784
• Main Authors: Sezgin, Erdinc, Levental, Ilya, Grzybek, Michal, Schwarzmann, Günter, Mueller, Veronika, Honigmann, Alf, Belov, Vladimir N., Eggeling, Christian, Coskun, Ünal, Simons, Kai, Schwille, Petra
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2012
• Subjects: Cell Membrane - chemistry; Cell Membrane - metabolism; Cholera Toxin - chemistry; Cholera Toxin - metabolism; Diffusion; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; G(M1) Ganglioside - chemistry; G(M1) Ganglioside - metabolism; GPMV; GUV; Ligands; Lipid; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Medicin och hälsovetenskap; Membrane Lipids - chemistry; Membrane Lipids - metabolism; Membrane Microdomains - chemistry; Membrane Microdomains - metabolism; Microscopy, Confocal; Nanotechnology; Partitioning; Protein Binding; Raft; Spectrometry, Fluorescence; STED-FCS; Unilamellar Liposomes - chemistry; Unilamellar Liposomes - metabolism; GUV; GPMV; Raft; Partitioning; STED-FCS; Lipid
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2012.03.007

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid–protein nanodomains, termed “rafts”. In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids (“raft lipids”, e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena. ►Raft lipid analogs show mispartitioning in both synthetic and cell-derived membrane systems. ►The partitioning of an analog is a function of type, size, polarity, charge and position of the dye. ►Acyl-chain labeling directly modulates bioactivity of lipids in a phase-specific manner. ►Partitioning in cell-derived GPMVs more closely follows raft predictions than in synthetic GUVs. ►Nanoscale diffusion in live cell membranes is uncorrelated with partitioning in model membranes.