Biomedical subjects

Gerald W Feigenson

Publications and source records attributed to Gerald W Feigenson.

10 recordsLinked to original sources

PubMed2007

Phase boundaries and biological membranes.

Bilayer mixtures of lipids are used by many researchers as chemically simple models for biological membranes. In particular, observations on three-component bilayer mixtures containing cholesterol show rich phase behavior, including several regions of two-phase coexistence and one region of three-phase coexistence. Yet, the relationship between these simple model mixtures and biological membranes, which contain hundreds of different proteins and lipids, is not clear. Many of the model mixtures have been chosen for study because they exhibit readily observed phase separations, not because they are good mimics of cell membrane components. If the many components of cell membranes could be grouped in some way, then understanding the phase behaviors of biological membranes might be enhanced. Furthermore, if the underlying interaction energies between lipids and proteins can be determined, then it might be possible to model the distributions of lipids and proteins in a bilayer membrane, even in complex mixtures.

Animals↗

PubMed2006

Phase behavior of lipid mixtures.

Biological membranes are two-dimensional mixtures of an enormous number of different components. Modeling cell membranes as simple bilayer mixtures reveals rich phase behavior, but how can we use the observed phase behavior to understand the real membranes?

Animals↗

PubMed2005

Phospholipid solubility determined by equilibrium distribution between surface and bulk phases.

A general strategy is proposed for determining the very low aqueous solubility limits of bilayer-forming phospholipids. The strategy exploits the inherent surface activity of phospholipids and has been termed EDSB, which stands for Equilibrium Distribution between Surface and Bulk phases. In this report, EDSB has been used to determine the critical bilayer concentration of dilauroylphosphatidylycholine (DLPC), a short-chain bilayer-forming phospholipid. At room temperature in neutral pH buffer, CBC(DLPC) = 2.5 x 10(-)(8) M. Using a mole fraction concentration scale, this corresponds to a standard-state free energy change of -12.8 kcal/mol for DLPC bilayer membrane formation.

Buffers↗

PubMed2005

Fluorescence methods to detect phase boundaries in lipid bilayer mixtures.

Phase diagrams of lipid mixtures can show several different regions of phase coexistence, which include liquid-disordered, liquid-ordered, and gel phases. Some phase regions are small, and some have sharp boundaries. The identity of the phases, their location in composition space, and the nature of the transitions between the phases are important for understanding the behavior of lipid mixtures. High fidelity phase boundary detection requires high compositional resolution, on the order of 2% compositional increments. Sample artifacts, especially the precipitation of crystals of anhydrous cholesterol, can occur at higher cholesterol concentrations unless precautions are taken. Fluorescence resonance energy transfer (FRET) can be used quantitatively to find the phase boundaries and even partition coefficients of the dyes between coexisting phases, but only if data are properly corrected for non-FRET contributions. Self-quenching of the dye fluorescence can be significant, distorting the data at dye concentrations that intuitively might be considered acceptable. Even more simple than FRET experiments, measurements of single-dye fluorescence can be used to find phase boundaries. Both FRET and single-dye fluorescence readily detect the formation of phase domains that are much smaller than the wavelength of light, i.e. "nanoscopic" domains.

Cholesterol↗

PubMed2005

Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells.

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Animals↗

PubMed2005

New method for determining tie-lines in coexisting membrane phases using spin-label ESR.

A full description of coexisting phases includes their respective compositions, which are provided by the thermodynamic tie-lines. Fluorescence microscopy enables visualization of coexisting lipid phases in giant unilamellar phases, but the composition information is missing. For cholesterol-containing lipid mixtures, knowledge of the compositions of the coexisting phases is important for understanding the nature of "membrane rafts". We propose and demonstrate a new method, based on ESR spectroscopy, for determining tie-lines in regions of two-phase coexistence in a ternary lipid mixture. Over 100 different lipid compositions containing the spin-labeled phospholipid 16-PC in or near the two-phase coexistence region of the liquid-disordered and the gel phases of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) were studied to determine five tie-lines, spread over virtually the full range of this coexistence region. The method is based on the facts that (1) along a tie-line the ESR spectrum must be a superposition of the two ESR spectra from the respective single phases at the phase boundaries (connected by the tie-line) in a ratio given by the lever rule; (2) along a tie-line the partition coefficient, K(p), for the spin-label, which is also determined in our method, must be constant. We do find that K(p) for 16-PC is close to unity, but its value depends on the particular tie-line. The coexisting phases in equilibrium are characterized by the K(p) of the spin-label and its respective dynamic parameters obtained from fitting the ESR spectra to dynamical models.

Cholesterol↗

PubMed2005

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy.

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2004

Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance.

The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2003

Nanoscopic lipid domain dynamics revealed by atomic force microscopy.

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.

1,2-Dipalmitoylphosphatidylcholine↗

PubMedDate not supplied

Atomic force microscopy of nanometric liposome adsorption and nanoscopic membrane domain formation.

Scanning probe microscopy studies of membrane fusion and nanoscopic structures were performed using hydrated single lipids and lipid mixtures. Extruded vesicles of DMPC and mixtures at various concentrations of DLPC, DPPC and cholesterol were deposited on freshly cleaved mica and studied in a fluid environment by AFM. The nanostructures formed by these extruded liposomes ranged from isolated unilamellar vesicles to flat sheet membranes and were marked influenced by thermodynamic phase behavior. For DMPC membrane, intact bilayers exhibited a phase transition process in agreement with large bilayer patches. In the DLPC, DPPC and cholesterol mixtures, nanoscopic domain diameters ranged from approximately 25 to 48nm with height differences of approximately 1.4nm; all values were lipid composition-dependent. Our data support and extend previous studies of microscopic domains and phase boundaries of the same mixtures in giant unilamellar vesicles determined by confocal light microscopy. Our approach for preparing and utilizing supported membrane structures is potentially relevant to studies of native cell membranes.

Adsorption↗