The liquid-ordered phase in membranes

A range of physiological processes has been imputed to lateral domain formation in biological membranes. However the molecular mechanisms of these functions and the details of how domain structures mediate these processes remain largely speculative. That domains exist in biomembranes and can be modeled in relatively simple lipid systems has contributed to our understanding of the principles governing phase behaviour in membranes. A presentation of these principles is the subject of this review. The condensing effect of sterols on phospholipids spread as monomolecular films at the air-water interface is described in terms of the dependence of the effect on sterol and phospholipid structure. The thermodynamics of sphingomyelin-cholesterol interactions are considered from calorimetric, densitometry and equilibrium cholesterol exchange measurements. Biophysical characterisation of the structure of liquid-ordered phase and its relationship with liquid-disordered phase is described from spectroscopic and X-ray scattering studies. Finally, the properties of liquid-ordered phase in the context of membrane physiology and permeability barrier properties are considered.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ∼10 °C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.     

Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy

By using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and curve fitting we have examined temperature dependence and composition dependence of the shape of the carbonyl band in phosphatidylcholine/cholesterol model membranes. Membranes were hydrated either in excess water or in excess deuterated water. The studied binary mixtures exhibit different lipid phases at appropriate temperature and amount of cholesterol, among them also the so-called liquid-ordered phase. The results confirm that cholesterol has a significant indirect influence on the carbonyl band through conformational and hydration effects. This influence was interpreted in view of the known temperature composition phase diagrams for inspected binary mixtures. In addition, direct interaction was observed, which could point to the presence of hydrogen bond between cholesterol and carbonyl group. This direct interaction, though weak, might play at least a partial role in the stabilization of cholesterol-rich lipid domains in model and biological membranes.     

Phase transition and lateral phase separation in planar lipid membranes as sensed by the lipophilic ion dipicrylamine

The permeation of the lipophilic ion dipicrylamine through planar lipid membranes formed from dipalmitoylphosphatidylcholine in n-decane shows an anomaly near the main phase transition of this system. Both the rate constant, k_i, of ion translocation across the membrane interior and the interfacial concentration, N, of this ion have a maximum at about 36°C. Analogous experiments were performed with tetraphenylborate. A considerably lesser effect of the phase transition was found. The addition of cholesterol leads to a broadening of the maxima for k_i and N. The time course of the current following a voltage jump shows a characteristic change below a temperature of about 45°C, if the molar ratio cholesterol/ phosphatidylcholine in the membrane forming solution exceeds 1. While the current transient decays exponentially above 45°C, a sum of two exponential terms yields an adequate fit below that temperature. This is regarded as evidence for a lateral phase separation below 45°C into structurally different domains, which provide two different pathways for dipicrylamine.     

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9-cis and 13-cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all-trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13-cis and 9-cis zeaxanthin, respectively; whereas, incorporation of all-trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all-trans < 9-cis ≤ 13-cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all-trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.     

Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts)

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 ∼ C6:0 ∼ C8:0 < no ceramide < C12:0 < C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 ∼ C6:0 < C2:0 < C12:0 ∼ no ceramide < C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.     

Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li⁺ among alkali cations, was found to be capable of mediating the transport of NH₄⁺ and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li⁺ (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH₄⁺ (0.05). The molecular factors involved in determining these selectivities are assessed.     

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.     

Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with ~5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.     

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by electron paramagnetic resonance spectroscopy

This paper continues the studies of vesicle-bound flavins (‘anisotropic flavin chemistry’). It is possible to anchor the flavin nucleus in various modes within the lipid/water interface by means of long aliphatic chains and using different saturated lipids, thereby mimicking the specific binding of the coenzyme to the apoprotein in flavoproteins. Based on absorption spectroscopy and EPR spectroscopy studies we explored the rotational mobility and the microenvironment of membrane-bound amphiflavin radicals. N(5)-unsubstiluted amphiflavin radicals exhibit a similarly high disproportionation constant as known from isotropic flavin chemistry. However, reasonable stabilization of the radical was achieved by introduction of an alkyl group in position 5 in the reduced state prior to the one-electron oxidation. Adopting the fine structure of the corresponding EPR spectra as assay for the mobility of the semiquinone, we determined rotational relaxation times ranging from 60 ns in the crystalline state down to 10 or 15 ns in the liquid-crystalline state of the membrane. The solvatochromic effect shown by absorption spectra of the membrane-bound flavin radicals reflects a dielectric constant of the microenvironment of ? = 30–40, corresponding to the lipid/water interface region. The results obtained in this study are consistent with those obtained previously, from fluorescence analyses, supporting our former conclusions.     

Sugars favour formation of hexagonal (HII) phase at the expense of lamellar liquid-crystalline phase in hydrated phosphatidylethanolamines

The disaccharides, sucrose and trehalose, markedly decreased (up to 17-13C°) the temperature of the lamellar to hexagonal (L_α →H_II) phase transition and simultaneously increase by 2–4 C° the temperature of the lamellar gel to lamellar liquid-crystal (L_β →L_α) phase transition in hydrated dihexadecylphosphatidylethanolamine and distearoylphosphatidylethanolamine. These two transitions merge and convert into a single L_β-H_II phase transition when dispersed in 2.4 M sucrose. These results are inconsistent with recent reports by (8) and (9)) which suggest that trehalose stabilizes the L_α phase relative to the H_II phase and shifts upwards beyond detectability the L_α-H_II transition. The present results are considered as a manifestation of the Hofmeister effect in which the sugars act as kosmotropic reagents stabilizing the structure of bulk water. This tends to decrease the area of contact between the lipid and the aqueous phases and favours the H_II and L_β phases relative to L_α phase. This hypothesis is consistent with the effects of chaotropic reagents on the L_α-H_II phase transition (Yeagle and Sen (1986) Biochemistry 25, 7518–7522) and on the stability of the lamellar phase of dipalmitoylphosphatidylcholine (Oku and MacDonald (1983) J. Biol. Chem. 258, 8733–8738).     

The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles

Time-resolved fluorescence anisotropy measurements on 1⁰-[4-(tri-methylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules in lipid vesicles of palmitoyloleoylphosphatidylcholine (POPC), PC extracted from egg yolk (EggPC), dioleoyl-PC (DOPC), dilinoleoyl-PC (DLPC), phosphatidylglycerol extracted from egg yolk (EggPG), dioleoyl-PG (DOPG), sulfoquinovosyldiacylglycerol (SQDG) and digalactosyl-DG (DGDG) with and without cholesterol are presented. The observed intensity decay curves are analyzed simultaneously in terms of the Brownian rotational diffusion model. The analysis thus yields the isotropic fluorescence decay, the initial anisotropy r (0), the order parameters P ₂ and P ₂ as well as the diffusion coefficient of the long molecular axis. It is shown that increasing unsaturation in the acyl chains of the PC lipids results in an increase in the rotational diffusion rates of the probes and a decrease in the order parameter P ₂. However, the value of P ₂ remains unchanged. The corresponding orientational distribution function of the probes is bimodal, with fractions lying preferentially parallel and perpendicular to the local vesicle surface. Surprisingly, the fraction of probe molecules lying with their long axes parallel to the bilayer surface increases with increasing unsaturation with a concomitant narrowing in the width of the distribution of the fraction lying perpendicular to it. As expected, cholesterol is found to increase the order parameters in all the systems and to suppress the tendency of the molecules to lie parallel to the bilayer surface. Furthermore, the rotational diffusion coefficients of the probes is found to increase in all the systems except for DLPC. Interestingly, the effects of unsaturation on the reorientational dynamics of TMA-DPH molecules in the vesicle systems are opposite to those found in the corresponding planar multibilayers (Deinum et al. 1988), whereas the same cholesterol effect is observed for the two systems. Nevertheless, the TMA-DPH molecules exhibit higher diffusion coefficients in the vesicle than in the planar multibilayer systems. In addition, a unimodal distribution of the probe molecules is found in the multibilayer systems. The differences between the two systems are ascribed to the differences in the radius of curvature and the hydration of the bilayers. Lastly we rationalize the bimodal distribution of the TMA-DPH molecules in the vesicles in terms of their observed partition between the lipid and aqueous phases.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[4-(trimethylammonio)-phenyl]-6-phenyl-1,3,5-hexatriene - POPC palmitoyloleoylphosphatidylcholine - EggPC PC extracted from egg yolk - DOPC dioleoyl-PC - DLPC dilinoleoyl-PC - EggPG phosphatidylglycerol extracted from egg yolk - DOPG dioleoyl-PG - SQDG sulfoquinovosyldiacylglycerol - DGDG di-galactosyl-DG - HPTLC high performance thin layer chromatography     

The effects of A23187 on the phospholipid phase transition of large unilamellar vesicles (LUVs) as detected by ultrasound spectroscopy

The effect of the hydrophobic Ca2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4: 1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG] membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, alpha lambda, was determined with a double crystal acoustic interferometer, as a function of temperature and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature (Tm) of the LUV suspensions remained near 42.0 degrees C, while the amplitude of alpha lambda at the phase transition increased dramatically. At Tm the relaxation frequency, where alpha lambda max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.     

The heterogeneous nuclear ribonucleoprotein (hnRNP) glorund functions in the Drosophila fat body to regulate lipid storage and transport

The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Our lab has shown that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. Here, we used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism.     

The effects of 2H2O on the phase transition of large unilamellar vesicle (LUV) suspensions as detected by ultrasound spectroscopy and electron spin resonance

The importance of water in the molecular dynamics of large unilamellar vesicle (LUV) suspensions, in which increasing portions of the water were replaced by 2H2O, was investigated. Determinations of the ultrasonic absorption coefficient per wavelength, alpha lambda, were performed as a function of temperature and frequency for LUVs (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine, DPPC, and dipalmitoylphosphatidylglycerol, DPPG) in the vicinity of their phospholipid phase transition, using a double crystal acoustic interferometer. Electron spin resonance (ESR) and differential scanning calorimetry (DSC) were also employed to probe this system. When increasing portions of the aqueous content of the LUV suspensions were replaced by 2H2O the phase transition temperature increased from 42.0 degrees C to 42.9 degrees C (indicating an increase in the activation energy of the transition), and the amplitude of alpha lambda at the phase transition increased. However, alpha lambda max as a function of frequency at the phase transition did not change with the addition of 2H2O, indicating that the relaxation time of the event responsible for the absorption of ultrasound was unaffected. The increase in the activation energy of the transition with the addition of 2H2O suggested that the mobility of phospholipids near the membrane/aqueous interface was changed. Electron spin resonance (ESR) experiments on LUVs with nitroxide spin probes positioned at the membrane/aqueous interface (5-doxyl stearate and CAT16) showed that LUVs in 2H2O have a broader splitting, Amax, at the membrane/aqueous interface than do LUVs in H2O. These results suggest that 2H2O changes the mobility and/or structure of the phospholipids in the region of the membrane/aqueous interface. This difference in Amax was not seen for the probe PC-12-doxyl stearate, which resides at the C-12 position of the bilayer.