Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin Mixture: Pulse-EPR Oxygen Probing

Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Biophysical Journal (Impact Factor: 3.97). 08/2011; 101(4):837-46. DOI: 10.1016/j.bpj.2011.07.014
Source: PubMed

ABSTRACT Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (l(d)) and liquid-ordered (l(o)) phases, and cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP). In the l(d) phase, coexisting with the l(o) phase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane. Changes in the OTP profile across the l(o) phase are complex. When the l(o) phase coexists with the l(d) phase, the OTP profile is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration (up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the CBD does not affect the OTP profile across the l(o) phase. OTP values in the CBD are significantly lower than in the l(o) phase.

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    • "Therefore it is reasonable to assume that GM1 is distributed between the two phases (when coexistence occurs) and leads on average to a high membrane order. As a control, we measured the GP temperature dependence for LUV lipid compositions typical of L d (pure egg PC, [54]) and pure L o (SM/Chol 50:50 mol/mol, [59]) phases with different GM1 amounts (Fig. 3B and C). As expected, at a given temperature, the GP values are much larger for vesicles in the L o phase than for those in the L d phase. "
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    ABSTRACT: Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including of C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and to a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing Lo nanodomains.
    Biochimica et Biophysica Acta (BBA) - Biomembranes 05/2014; 1838(8). DOI:10.1016/j.bbamem.2014.05.002 · 3.84 Impact Factor
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    • "lipid bilayer membranes used in this work were multilamellar dispersions (multilamellar liposomes) made from the lipid extracts from either the porcine eye-lens cortex or nucleus and containing ~1 mol% spin label (n-PC, 9-SASL, ASL, or CSL). The membranes were prepared using the film deposition method described previously (Kusumi et al., 1986; Mainali et al., 2011c). "
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    ABSTRACT: The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eye lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes, because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes-namely, the domain formed by boundary lipids and the domain formed by trapped lipids-were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.
    Experimental Eye Research 02/2012; 97(1):117-29. DOI:10.1016/j.exer.2012.01.012 · 2.71 Impact Factor
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    • "When the Chol content reaches saturation, the most drastic changes that occur in the bilayer are observed in the profiles of hydrophobicity and the oxygen transport parameter [18] [19]. In liquid-ordered PC bilayers of the lowest Chol content of ~30 mol%, as well as in bilayers without Chol, these profiles are bell-shaped [20] [21]. At Chol saturation , the shape of these profiles becomes rectangular and is practically the same as for lens lipid membranes [22] [23]. "
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    ABSTRACT: Molecular dynamics (MD) simulations of a mono-cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer and a POPC bilayer containing 50mol% cholesterol (POPC-Chol50) were carried out for 200ns to compare the spatial organizations of the pure POPC bilayer and the POPC bilayer saturated with Chol. The results presented here indicate that saturation with Chol significantly narrows the distribution of vertical positions of the center-of-mass of POPC molecules and POPC atoms in the bilayer. In the POPC-Chol50 bilayer, the same moieties of the lipid molecules are better aligned at a given bilayer depth, forming the following clearly separated membrane regions: the polar headgroup, the rigid core consisting of steroid rings and upper fragments of the acyl chains, and the fluid hydrocarbon core consisting of Chol chains and the lower fragments of POPC chains. The membrane surface of the POPC-Chol50 bilayer is smooth. The results have biological significance because the POPC-Chol50 bilayer models the bulk phospholipid portion of the fiber-cell membrane in the eye lens. It is hypothesized that in the eye lens cholesterol-induced smoothing of the membrane surface decreases light-scattering and helps to maintain lens transparency.
    Biochimica et Biophysica Acta 10/2011; 1818(3):520-9. DOI:10.1016/j.bbamem.2011.10.023 · 4.66 Impact Factor
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