RESUMEN
Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly because of the activation of enzymes like acid sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid-ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), too, is present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid-ordered phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains rich in Cer coexist with the continuous fluid phase of the membrane. Here, we studied the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mole ratios were 3:1, 3:2, and 3:3, at a fixed 7:3 phospholipid:cholesterol mol ratio. Both PSM and PCer were monitored in separate samples for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Moreover, the effect of replacing PSM with DPPC was investigated to test the impact on membrane phase behavior of replacing the sphingosine with a palmitoylated glycerol backbone. We found that PCer can increase acyl chain order in both PSM:Chol and DPPC:Chol bilayers. Especially in bilayers with Chol:PCer 1:1 molar ratios, PCer induces highly stable So phase domains in both PSM and DPPC bilayers near 37°C. However, PCer has a more pronounced ordering effect on PSM compared to DPPC bilayers.
Asunto(s)
Ceramidas/química , Colesterol/química , Deuterio/química , Espectroscopía de Resonancia Magnética , Fosfolípidos/química , TemperaturaRESUMEN
In this paper we investigate the pseudo-ternary phase diagram of glycerol monooleate (GMO), a cationic lipid (DOTAP - 1,2-dioleoyl-3-trimethylammonium propane), and a "PEGylated" lipid (DOPE-PEG - 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 kDa]) in excess water. We use small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-EM) to map out a phase diagram in a regime of low DOPE-PEG content (1-5 mol%), which is pertinent for the application of lipid systems as carriers of biomolecular cargo to cells. Pure GMO is known to self-assemble into bicontinuous cubic phases of the gyroid type at low water content and of the diamond type in excess water. These complex structures have numerous advantages reaching beyond drug delivery, e.g. as protein crystallization matrices, but their formulation is challenging as very small contents of guest molecules can shift the phase behavior towards other geometries such as the lamellar phase. In this work, we show that the ternary GMO/DOTAP/DOPE-PEG system allows the stabilization of bicontinuous cubic phases in excess water over a wide composition range. The symmetry of the phase can be tuned by varying the amount of PEGylated lipid, with the primitive type dominating at low DOPE-PEG content (1-3 mol%) and the diamond phase arising at 5 mol% DOPE-PEG. In addition, we found that the diamond phase is virtually non-responsive to electrostatic swelling. In contrast, primitive bicontinuous cubic lattice dimensions swell up in equilibrium to 650 Å with increased cationic lipid content.
RESUMEN
The hierarchical assembly of lipids, as modulated by composition and environment, plays a significant role in the function of biological membranes and a myriad of diseases. Elevated concentrations of calcium ions and cardiolipin (CL), an anionic tetra-alkyl lipid found in mitochondria and some bacterial cell membranes, have been implicated in pneumonia recently. However, their impact on the physicochemical properties of lipid assemblies in lungs and how it impairs alveoli function is still unknown. We use small- and wide-angle X-ray scattering (S/WAXS) and solid-state nuclear magnetic resonance (ssNMR) to probe the structure and dynamics of lung-mimetic multilamellar bodies (MLBs) in the presence of Ca2+ and CL. We conjecture that CL overexpressed in the hypophase of alveoli strongly affects the structure of lung-lipid bilayers and their stacking in the MLBs. Specifically, S/WAXS data revealed that CL induces significant shrinkage of the water-layer separating the concentric bilayers in multilamellar aggregates. ssNMR measurements indicate that this interbilayer tightening is due to undulation repulsion damping as CL renders the glycerol backbone of the membranes significantly more static. In addition to MLB dehydration, CL promotes intrabilayer phase separation into saturated-rich and unsaturated-rich lipid domains that couple across multiple layers. Expectedly, addition of Ca2+ screens the electrostatic repulsion between negatively charged lung membranes. However, when CL is present, addition of Ca2+ results in an apparent interbilayer expansion likely due to local structural defects. Combining S/WAXS and ssNMR on systems with compositions pertinent to healthy and unhealthy lung membranes, we propose how alteration of the physiochemical properties of MLBs can critically impact the breathing cycle.
Asunto(s)
Membrana Celular/química , Membrana Celular/efectos de los fármacos , Neumonía/fisiopatología , Calcio/farmacología , Cardiolipinas/farmacología , Humanos , Membrana Dobles de Lípidos/química , Pulmón/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Dispersión de Radiación , Rayos XRESUMEN
Fluorescence microscopy is an important technique for studying lipid membranes and is increasingly being used for examining liquid ordered-liquid disordered phase coexistence. Liquid-liquid phase coexistence is a phenomenon of biological interest because it led to the lipid raft hypothesis, which postulates the existence of lateral heterogeneities in cell membranes. Observation of membrane heterogeneity relies on differential distribution of fluorescent membrane markers, but this can also modify the phase behavior, complicating the observation. We have used 2H NMR to measure the physical changes to 35:35:30 (mol/mol) DOPC/DPPC-D62/chol membranes introduced by fluorescent probes Laurdan and naphthopyrene. We measured miscibility transition temperature (Tmix) and DPPC-D62 chain order for a range of probe concentrations. We found that up to 0.5 mol% of the equipartitioning probe Laurdan does not influence DPPC-D62 acyl chain order or phase behavior. In contrast, 2.0 mol% Laurdan slightly increases the fraction of DPPC-D62 in the liquid disordered phase below the Tmix and increases Tmix by 1 °C. Conversely, the nominally liquid ordered phase preferring probe naphthopyrene slightly perturbs the membrane even at concentrations as low as 0.3 mol%. This suggests that the strength of fluorescent probe partitioning between liquid ordered and liquid disordered phases correlates with the degree of perturbation to membrane phase behavior.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Colorantes Fluorescentes/química , Fosfatidilcolinas/química , Microscopía Fluorescente , Temperatura de TransiciónRESUMEN
Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane's physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by ²H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our ²H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids' miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer.
Asunto(s)
Ceramidas/química , Transición de Fase , Esfingomielinas/química , Colesterol/química , Cristales Líquidos/química , TemperaturaRESUMEN
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.