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Biomedical subjects

Sarah L Veatch

Publications and source records attributed to Sarah L Veatch.

10 recordsLinked to original sources
PubMed2007

Fluorescent probes alter miscibility phase boundaries in ternary vesicles.

We use 2H NMR to study the effects of probes on the miscibility transition in multilamellar vesicles of di(18:1) phosphatidylcholine (PC; DOPC), chain perdeuterated di(16:0)PC (DPPCd62), and cholesterol both with and without 0.5 mol % of the fluorescent probes DiIC12 and DiOC18. Both probes raise the miscibility transition temperature in dispersions of 1:1 DOPC/DPPCd62 + 30% cholesterol but to differing extents. In membranes containing the popular probe DiIC12, the fraction of DPPCd62 lipids in the liquid disordered phase is increased, and the ordering of that phase is reduced even at low temperatures. All findings are consistent with a probe-induced expansion of the entire miscibility phase boundary. We examine membranes with smaller DiIC12 fractions and find a significant increase in transition temperature for samples with 0.05 mol % DiIC12, demonstrating that trace components can dramatically alter membrane phase behavior.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Closed-loop miscibility gap and quantitative tie-lines in ternary membranes containing diphytanoyl PC.

Vesicles containing ternary mixtures of diphytanoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and cholesterol produce coexisting liquid phases over an unusually large range of temperature and composition. Liquid domains persist well above the DPPC chain melting temperature (41 degrees C), resulting in a closed-loop miscibility gap bounded by two critical points at fixed temperature. Quantitative tie-lines are determined directly from 2H NMR spectra using a novel analysis, and are found to connect a liquid-disordered phase rich in diphytanoyl PC with a liquid-ordered phase rich in DPPC. The direction of the tie-lines implies that binary DPPC/cholesterol mixtures are in one uniform phase above 41 degrees C. All 2H NMR results for tie-lines are verified by independent fluorescence microscopy results.

Cholesterol↗
PubMed2005

Seeing spots: complex phase behavior in simple membranes.

Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes.

Cell Membrane↗
PubMed2005

Sterol structure determines miscibility versus melting transitions in lipid vesicles.

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

Miscibility phase diagrams of giant vesicles containing sphingomyelin.

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2004

Nonequilibrium behavior in supported lipid membranes containing cholesterol.

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Schäfer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.

Air↗
PubMed2003

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol.

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Organization in lipid membranes containing cholesterol.

A fundamental attribute of raft formation in cell membranes is lateral separation of lipids into coexisting liquid phases. Using fluorescence microscopy, we observe spontaneous lateral separation in free-floating giant unilamellar vesicles. We record coexisting liquid domains over a range of composition and temperature significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixtures that produce liquid domains in bilayer membranes produce two upper miscibility critical points in the phase diagrams of monolayers.

Cell Membrane↗
PubMed2002

On the binding preference of human groups IIA and X phospholipases A2 for membranes with anionic phospholipids.

Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

Anions↗