Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy

Abstract

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI-C 18 ) was excluded from sphingomyelinenriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholineenriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.More than 10 years ago, the hypothesis was formulated that cellular membranes are organized in discrete dynamic entities, called lipid rafts (1, 2). Studies on epithelial cell polarity revealed that lipids, in particular sphingolipids and cholesterol, were laterally organized in the exoplasmic leaflet of the apical plasma membrane according to a variable short and long range order. Furthermore, distinct proteins were shown to selectively partition into lipid rafts, indicating that rafts could serve as specific sites for molecular sorting and polarized transport.

Glycans pattern the phase behaviour of lipid membranes

“…We also find that glycan-patterned phase separation is thermally reversible -thus indicating that the effect is thermodynamic rather than kinetic -and that phase patterning results likely due to a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes [6][7][8] , potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes [9][10][11][12][13] , and may indicate a more intimate coupling between cellular lipidomes and glycomes than realized currently.…”

“…In general, diffusion of fluorescent probes is about 1-10 times slower in the Lo phase compared to the Ld phase due to the increased viscosity of the ordered phase 10 . Lipids are immobile in the So phase 10 .…”

“…Indeed, while there are many reports of lipid membranes supported on non-biological polymers such as polyethylene glycol, polyacrylamide or polyethyleneimine [17][18][19][20] which seek to mitigate non-'equilibrium' behaviour, such systems have been used mainly to study the effects of polymer hydration 21 on the mobility of lipids 18,19 , or to preserve the function of transmembrane protein inclusions 17,22 . Furthermore, while it is widely acknowledged that a greater understanding of parameters that influence the size and length-scale of membrane domains is required [6][7][8][9][10][11][12][13]23,24 , as far as we know, systematic studies exploring the effects of biopolymers (the subject of this letter), or other more commonly used polymers in the field [17][18][19][20] on the phase behaviour of lipid membranes have not been conducted. To investigate the possible effects of biologically relevant polymers on the behaviour of membranes, we designed an in vitro solid-supported experimental platform that allows, through fluorescent labelling and confocal microscopy, the study of lipid membranes interacting with hydrated networks of glycans with arbitrary glycan composition and variable network configurations.…”

Agonist mobility on supported lipid bilayers affects Fas mediated death response

“…For example, reducing membrane fluidity by addition of cholesterol reduced the diffusion coefficients of lipid and protein to the same degree. Thus, streptavidin or agonist binding has, if at all, only a small effect on mobility, which can be explained by the negligible hydrodynamic drag introduced by the protruding protein domains in the aqueous phase [26,39].…”

Protective Effects of Glycyrrhizin against .BETA.2-Adrenergic Receptor Agonist-Induced Receptor Internalization and Cell Apoptosis

“…21) Recently, Sieben et al 22) found that pre-incubation for 24 h with the saponin compound R-hed-erin could inhibit the terbutaline-stimulated internalization of the b 2 AR, which is in agreement with the fact that saponins are cholesterol-complexing agents and that cholesterol depletion is known to inhibit receptor internalization. [23][24][25][26] Chemically, glycyrrhizin is a triterpenoid saponin glycoside, therefore, we hypothesized that glycyrrhizin may have a positive effect on the regulation of b 2 AR agonist-induced internalization of b 2 ARs.…”