title: 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.
authors.affiliation: Biophysics/BIOTEC, TU Dresden. Tatzberg 47-51, 01307 Dresden, Germany.
authors.forename: Erdinc
authors.initials: E
authors.lastname: Sezgin
authors.forename: Ilya
authors.initials: I
authors.lastname: Levental
authors.forename: Michal
authors.initials: M
authors.lastname: Grzybek
authors.forename: Günter
authors.initials: G
authors.lastname: Schwarzmann
authors.forename: Veronika
authors.initials: V
authors.lastname: Mueller
authors.forename: Alf
authors.initials: A
authors.lastname: Honigmann
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Belov
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
authors.forename: Unal
authors.initials: U
authors.lastname: Coskun
authors.forename: Kai
authors.initials: K
authors.lastname: Simons
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002772:Cholera Toxin / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D036103:Nanotechnology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D008051:Lipid Bilayers; D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D005677:G(M1) Ganglioside; D002772:Cholera Toxin