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

Abstract

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 GMI 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.

Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes

“…Förster resonance energy transfer (FRET) 122 is able to probe much smaller distances and has proven extremely useful for studying domain formation and phase behavior 123, 124 as well as identifying raft associating molecules 125 . FM has been also been used to study partitioning, diffusion and ligand binding of raft lipid analogs in model and cellular plasma membranes 126 . Fluorescence recovery after photobleaching (FRAP) 127 is a particularly useful approach for studying lateral diffusion in bilayers 128 and distinguishing the liquid ordered (Lo) phase in raft forming models 129,130 .…”

Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond

“…CT-and LT-binding result in the clustering of GM1 gangliosides, targeting more GM1 for retrograde transport to the ER. Both lipid sorting and toxin binding depend on lipid structure and membrane fluidity, resulting in a delicately balanced interaction network where care must be taken when performing analyses to not disturb the system under investigation [115,116]. For example, while CTB has been shown to bind preferably to ordered lipid phases [115,117], attachment of a fluorescent tag to the GM1 ganglioside partitioned CTB to the liquid disordered phase [116].…”

The Stability of Lipid Rafts-Like Micro-Domains Is Dependent on the Available Amount of Cholesterol

“…The viability of the cells was restored upon addition of cholesterol. On the other hand, depletion of cholesterol caused lipid raft disruption, and as a consequence lipid rafts were rendered nonfunctional [8]- [10]. Hence, a clear correlation seems to exist between the number of viable lipid rafts in cell membranes and the amount of available cholesterol.…”

“…Observations using atomic force microscopy demonstrated that ethanol influenced the bilayer properties according to membrane lateral organization [24]. Calorimetric investigations of mixtures of ethanol and liposomes were also performed, and the results obtained showed that the addition of cholesterol in lower concentrations helped to increase the interactions, while cholesterol in higher concentrations helped reduce the interactions [8] [25].…”