The Experts below are selected from a list of 1287 Experts worldwide ranked by ideXlab platform
Deborah A Brown - One of the best experts on this subject based on the ideXlab platform.
-
Preparation of detergent-resistant membranes (DRMs) from cultured mammalian cells.
Methods of Molecular Biology, 2014Co-Authors: Deborah A BrownAbstract:: Detergent-resistant membranes (DRMs) isolated from cells are enriched in proteins and lipids with a high affinity for lipid rafts, or membrane microdomains in the Liquid-Ordered Phase. Enrichment in DRMs provides a good indication that a protein is "raftophilic," and may be present in rafts in cell membranes before extraction. Here, I describe preparation of Triton X-100-insoluble DRMs from cultured mammalian cells on sucrose gradients. Methods for analyzing the distribution of particular proteins across the gradient, and for recovering DRMs for further use are presented.
-
Structure and function of membrane rafts.
International Journal of Medical Microbiology, 2004Co-Authors: Deborah A BrownAbstract:Abstract Lipids do not always mix uniformly in membranes, but can cluster to form microdomains. We will consider one type of microdomain that can form in cell membranes. These are enriched in cholesterol and sphingolipids, and are referred to as rafts. Rafts probably exist in membranes in the Liquid-Ordered Phase or a Phase with similar properties. We will briefly review membrane lipid Phase behavior, and the differences between Liquid-crystalline, Liquid-Ordered, and gel-Phase membrane bilayer domains. We will present evidence suggesting that phospholipid-rich, Liquid-crystalline Phase domains and sphingolipid-rich, Liquid-Ordered Phase domains (rafts) can exist in equilibrium in biological membranes, especially the plasma membrane. Preferential partitioning of membrane proteins into rafts can affect function. Among the proteins that are targeted to rafts are those anchored in the outer leaflet of the membrane through covalent attachment to a special glycolipid, glycosyl phosphatidylinositol (GPI). Other proteins that are linked to saturated acyl chains, such as those that are directly acylated with two or more palmitate chains, or a palmitate and a myristate chain, are also targeted to rafts. Targeting of GPI-anchored proteins and other proteins to rafts plays a role in signal transduction in hematopoietic cells, and possibly also in sorting in intracellular membranes and regulation of cell-surface proteolysis in other mammalian cells.
-
role of lipid modifications in targeting proteins to detergent resistant membrane rafts many raft proteins are acylated while few are prenylated
Journal of Biological Chemistry, 1999Co-Authors: Karin A Melkonian, Anne G Ostermeyer, James Z Chen, Michael Roth, Deborah A BrownAbstract:Abstract Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the Liquid-Ordered Phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an Ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Madin-Darby canine kidney cell DRMs (other than cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza hemagglutinin with DRMs required all three of its palmitoylated Cys residues. Prenylated proteins, detected by [3H]mevalonate labeling or by blotting for Rap1, Rab5, Gβ, or Ras, were excluded from DRMs. Rab5 and H-Ras each contain more than one lipid group, showing that hydrophobicity alone does not target multiply lipid-modified proteins to DRMs. Partitioning of covalently linked saturated acyl chains into Liquid-Ordered Phase domains is likely to be an important mechanism for targeting proteins to DRMs.
-
on the origin of sphingolipid cholesterol rich detergent insoluble cell membranes physiological concentrations of cholesterol and sphingolipid induce formation of a detergent insoluble Liquid Ordered lipid Phase in model membranes
Biochemistry, 1997Co-Authors: Sharmin N Ahmed, Deborah A Brown, Erwin LondonAbstract:Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the Liquid-Ordered Phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130−12134]. In order to detect formation of the Liquid-Ordered Phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the Phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel Phase coexisted with a 12SLPC-enriched Liquid-disOrdered fluid Phase at 23 °C. As expected, Phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid Phase was present. Including 33 mol % cholesterol in ...
-
On the Origin of Sphingolipid/Cholesterol-Rich Detergent-Insoluble Cell Membranes: Physiological Concentrations of Cholesterol and Sphingolipid Induce Formation of a Detergent-Insoluble, Liquid-Ordered Lipid Phase in Model Membranes
Biochemistry, 1997Co-Authors: Sharmin N Ahmed, Deborah A Brown, Erwin LondonAbstract:Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the Liquid-Ordered Phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130−12134]. In order to detect formation of the Liquid-Ordered Phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the Phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel Phase coexisted with a 12SLPC-enriched Liquid-disOrdered fluid Phase at 23 °C. As expected, Phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid Phase was present. Including 33 mol % cholesterol in ...
Peter J Quinn - One of the best experts on this subject based on the ideXlab platform.
-
A lipid matrix model of membrane raft structure.
Progress in Lipid Research, 2010Co-Authors: Peter J QuinnAbstract:Domains in cell membranes are created by lipid–lipid interactions and are referred to as membrane rafts. Reliable isolation methods have been developed which have shown that rafts from the same membranes have different proteins and can be sub-fractionated by immunoaffinity methods. Analysis of these raft subfractions shows that they are also comprised of different molecular species of lipids. The major lipid classes present are phospholipids, glycosphingolipids and cholesterol. Model studies show that mixtures of phospholipids, particularly sphingomyelin, and cholesterol form Liquid-Ordered Phase with properties intermediate between a gel and fluid Phase. This type of Liquid-Ordered Phase dominates theories of domain formation and raft structure in biological membranes. Recently it has been shown that sphingolipids with long (22–26C) N-acyl fatty acids form quasi-crystalline bilayer structures with diacylphospholipids that have well-defined stoichiometries. A two tier heuristic model of membrane raft structure is proposed in which Liquid-Ordered Phase created by a molecular complex between sphingolipids with hydrocarbon chains of approximately equal length and cholesterol acts as a primary staging area for selecting raft proteins. Tailoring of the lipid anchors of raft proteins takes place at this site. Assembly of lipid-anchored proteins on a scaffold of sphingolipids with asymmetric hydrocarbon chains and phospholipids arranged in a quasi-crystalline bilayer structure serves to concentrate and orient the proteins in a manner that couples them functionally within the membrane. Specificity is inherent in the quasi-crystalline lipid structure of Liquid-Ordered matrices formed by both types of complex into which protein lipid anchors are interpolated. An interaction between the sugar residues of the glycolipids and the raft proteins provides an additional level of specificity that distinguishes one raft from another.
-
long n acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form Liquid Ordered Phase
Biochimica et Biophysica Acta, 2009Co-Authors: Peter J QuinnAbstract:Abstract The structure and thermotropic Phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2°/min between 25 and 85 °C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar Phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 °C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel Phase phospholipid to the fluid Phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of Phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a Phase transition of pure phospholipid at about 43 °C coexisting with a Liquid-Ordered Phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 °C which showed that a glucolipid-rich Phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich Phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 °C with pure phospholipid in gel Phase and 42:58 mole ratio at 65 °C when the phospholipid was in the fluid Phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.
-
Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol.
Biochimica et Biophysica Acta, 2009Co-Authors: Peter J Quinn, Claude WolfAbstract:Abstract The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20° and 50 °C using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20° and 50 °C. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as Liquid-Ordered Phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 °C the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel Phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disOrdered hydrocarbon in dispersions of pure sphingomyelin and as Liquid-Ordered Phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel Phases of egg and brain sphingomyelins at 20 °C. The gel Phases coexist with Liquid-Ordered Phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel Phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened Phase transition endotherm originates from dilution of the cholesterol-rich Liquid-Ordered Phase by mobilization of sphingomyelin from the melting gel Phase.
-
The role of sterol rings and side chain on the structure and Phase behaviour of sphingomyelin bilayers.
Molecular Membrane Biology, 2009Co-Authors: Peter J Quinn, Zhiwu YuAbstract:The role of the side chain of sterols and the sterol ring structure on the formation of Ordered Phases of the type observed in membrane rafts has been examined in aqueous dispersions of binary mixtures of sphingomyelin and androsterol. Comparisons have been made with binary systems of cholesterol, stigmasterol, b-sitosterol, and ergosterol with either sphingomyelin or dipalmitoylphosphatidylcholine. Thermotropic Phase behaviour and structure of the mixed aqueous dispersions were characterized by differential scanning calorimetry, synchrotron X-ray diffraction, freeze-fracture electron microscopy, and Fourier-transform infrared spectroscopy. We show that: (i) Androsterol is less efficient in promoting the formation of Liquid-Ordered Phase than other naturally occurring sterols which possess a side chain, (ii) cholesterol is the most efficient sterol of those investigated in forming Liquid-Ordered Phase, (iii) the molecular stoichiometry of egg sphingomyelin and androsterol in the Liquid-Ordered Phase is about 2:1, and (iv) sphingomyelin can form more stable Liquid-Ordered Phase than glycerophospholipid in binary systems containing androsterol.
-
the Liquid Ordered Phase in membranes
Biochimica et Biophysica Acta, 2009Co-Authors: Peter J Quinn, Claude WolfAbstract: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.
Erwin London - One of the best experts on this subject based on the ideXlab platform.
-
on the origin of sphingolipid cholesterol rich detergent insoluble cell membranes physiological concentrations of cholesterol and sphingolipid induce formation of a detergent insoluble Liquid Ordered lipid Phase in model membranes
Biochemistry, 1997Co-Authors: Sharmin N Ahmed, Deborah A Brown, Erwin LondonAbstract:Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the Liquid-Ordered Phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130−12134]. In order to detect formation of the Liquid-Ordered Phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the Phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel Phase coexisted with a 12SLPC-enriched Liquid-disOrdered fluid Phase at 23 °C. As expected, Phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid Phase was present. Including 33 mol % cholesterol in ...
-
On the Origin of Sphingolipid/Cholesterol-Rich Detergent-Insoluble Cell Membranes: Physiological Concentrations of Cholesterol and Sphingolipid Induce Formation of a Detergent-Insoluble, Liquid-Ordered Lipid Phase in Model Membranes
Biochemistry, 1997Co-Authors: Sharmin N Ahmed, Deborah A Brown, Erwin LondonAbstract:Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the Liquid-Ordered Phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130−12134]. In order to detect formation of the Liquid-Ordered Phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the Phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel Phase coexisted with a 12SLPC-enriched Liquid-disOrdered fluid Phase at 23 °C. As expected, Phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid Phase was present. Including 33 mol % cholesterol in ...
Steven L Regen - One of the best experts on this subject based on the ideXlab platform.
-
effects of isoflurane halothane and chloroform on the interactions and lateral organization of lipids in the Liquid Ordered Phase
Langmuir, 2011Co-Authors: Serhan Turkyilmaz, Paulo F. Almeida, Steven L RegenAbstract:The first quantitative insight has been obtained into the effects that volatile anesthetics have on the interactions and lateral organization of lipids in model membranes that mimic "lipid rafts". Specifically, nearest-neighbor recogntion measurements, in combination with Monte Carlo simulations, have been used to investigate the action of isoflurane, halothane, and chloroform on the compactness and lateral organization of cholesterol-rich bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the Liquid-Ordered (l(o)) Phase. All three anesthetics induce a similar weakening of sterol-phospholipid association, corresponding to ca. 30 cal/mol of lipid at clinically relevant concentrations. Monte Carlo lattice simulations show that the lateral organization of the l(o) Phase, under such conditions, remains virtually unchanged. In sharp contrast to their action on the l(o) Phase, these anesthetics have been found to have a similar strengthening effect on sterol-phospholipid association in the Liquid-disOrdered (l(d)) Phase. The possibility of discrete complexes being formed between DPPC and these anesthetics and the biological relevance of these findings are discussed.
-
oxysterol induced rearrangement of the Liquid Ordered Phase a possible link to alzheimer s disease
Journal of the American Chemical Society, 2009Co-Authors: Hideyuki Mitomo, Wenhua Chen, Steven L RegenAbstract:Nearest-neighbor recognition measurements have been made for an exchangeable phospholipid (A) interacting with an exchangeable form of cholesterol (B) in host membranes derived from 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations of cholesterol, 7β-hydroxycholesterol (7β-OH), and 25-hydroxycholesterol (25-OH). Whereas partial replacement of cholesterol with 7β-OH strengthens the association between A and B, a similar substitution with 25-OH weakens this association. A model that accounts for this dichotomy, and the possible relevance of these findings to the cytotoxicity of 7β-OH and to Alzheimer’s disease are briefly discussed.
-
cholesterol phospholipid association in fluid bilayers a thermodynamic analysis from nearest neighbor recognition measurements
Biophysical Journal, 2006Co-Authors: Jianbing Zhang, Bingwen Jing, Paulo F. Almeida, Steven L RegenAbstract:The mixing behavior of exchangeable, disulfide-based mimics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been examined as a function of temperature in host membranes made from DPPC and cholesterol in the Liquid-disOrdered Phase (ld), in the Liquid-Ordered Phase (lo), and in the Liquid-disOrdered/Liquid-Ordered coexistence region (ld/lo). In the ld region, lipid mixing was found to be temperature insensitive, reflecting close to ideal behavior. In contrast, a significant temperature dependence was observed in the lo Phase from 45 to 60°C, when 35 or 40 mol % sterol was present. In this region, sterol-phospholipid association was characterized by ΔHo = −2.06 ± 0.14 kcal/mol of phospholipid and ΔS° = −4.48 ± 0.44 cal/K mol of phospholipid. From 60 to 65°C, the mixing of these lipids was found to be insensitive to temperature, and sterol-phospholipid association was now entropy driven; that is, ΔHo = −0.23 ± 0.38 kcal/mol of phospholipid and ΔS° = +1.68 ± 1.12 cal/K mol of phospholipid. In the Liquid-disOrdered/Liquid-Ordered coexistence region, changes in lipid mixing reflect changes in the Phase composition of the membrane.
-
ethanol induced reorganization of the Liquid Ordered Phase enhancement of cholesterol phospholipid association
Journal of the American Chemical Society, 2006Co-Authors: Jianbing Zhang, Bingwen Jing, Steven L RegenAbstract:This paper records what is believed to be the first evidence for the reorganization of the Liquid-Ordered Phase by ethanol. Specifically, ethanol has been found to significantly enhance sterol−phospholipid association in Liquid-Ordered bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) plus cholesterol and also 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) plus cholesterol. The evidence for such reorganization comes from a series of nearest-neighbor recognition (NNR) experiments that have been carried out, where low concentrations of equilibrating lipid dimers (i.e., “reporter molecules”) have been used to detect changes in the Phase composition of host membranes made from varying mixtures of DPPC/cholesterol, and also DSPC/cholesterol, in the presence and in the absence of ethanol. These findings have important biological implications, which are briefly discussed.
-
Ethanol-Induced Reorganization of the Liquid-Ordered Phase: Enhancement of Cholesterol−Phospholipid Association
Journal of the American Chemical Society, 2006Co-Authors: Jianbing Zhang, Bingwen Jing, Steven L RegenAbstract:This paper records what is believed to be the first evidence for the reorganization of the Liquid-Ordered Phase by ethanol. Specifically, ethanol has been found to significantly enhance sterol−phospholipid association in Liquid-Ordered bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) plus cholesterol and also 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) plus cholesterol. The evidence for such reorganization comes from a series of nearest-neighbor recognition (NNR) experiments that have been carried out, where low concentrations of equilibrating lipid dimers (i.e., “reporter molecules”) have been used to detect changes in the Phase composition of host membranes made from varying mixtures of DPPC/cholesterol, and also DSPC/cholesterol, in the presence and in the absence of ethanol. These findings have important biological implications, which are briefly discussed.
Masahito Yamazaki - One of the best experts on this subject based on the ideXlab platform.
-
Water permeability of lipid membranes of GUVs and its dependence on actin cytoskeletons inside the GUVs
2008 International Symposium on Micro-NanoMechatronics and Human Science, 2008Co-Authors: Takuya Yoshitani, Masahito YamazakiAbstract:Response of cells to osmotic pressure and control of water permeability of biomembranes are very important for cells to survive. There have been many studies using cells, but their details remain unknown. In this study, we investigated water permeability of various lipid membranes of GUVs by the micropipet aspiration technique, and then its dependence on actin cytoskeletons inside the GUVs. First, we measured of water permeability of various lipid membranes. When we transferred a single GUV fixed by a micropipet using a small aspiration pressure into a buffer with a higher osmolarity, the volume of the GUV decreased due to the water efflux. We determined the decrease in the GUV volume by the increase in aspiration length in the micropipet. We analyzed quantitatively the time course of volume change of the GUV on the basis of the theoretical equation, and obtain water permeability of the lipid membrane. The average value of water permeability of DOPC membrane in the L α Phase 45 ± 4 μm/s. The water permeability depended greatly on kinds of lipid membranes. For example, the water permeability of DPPC/cholesterol (6/4, molar ratio) membrane in a Liquid-Ordered Phase was 1.3 ± 0.1 μm/s, which was much smaller than that of DOPC membrane. Second, we investigated the effect of actin filaments on water permeability of lipid membranes. We succeeded in preparing DOPC/DOPG (1/1)-GUVs containing 1 mg/mL actin filaments.
-
vesicle fission of giant unilamellar vesicles of Liquid Ordered Phase membranes induced by amphiphiles with a single long hydrocarbon chain
Langmuir, 2007Co-Authors: Yasuyuki Inaoka, Masahito YamazakiAbstract:Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/cholesterol membranes (6/4) in the Liquid-Ordered (lo) Phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolat...
-
stability of giant unilamellar vesicles and large unilamellar vesicles of Liquid Ordered Phase membranes in the presence of triton x 100
Biochimica et Biophysica Acta, 2004Co-Authors: Yukihiro Tamba, Tomoki Tanaka, Takeshi Yahagi, Yuko Yamashita, Masahito YamazakiAbstract: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.
-
shape changes and vesicle fission of giant unilamellar vesicles of Liquid Ordered Phase membrane induced by lysophosphatidylcholine
Langmuir, 2004Co-Authors: Tomoki Tanaka, Ryoko Sano, And Yuko Yamashita, Masahito YamazakiAbstract:Liquid-Ordered Phase (lo Phase) of lipid membranes has properties that are intermediate between those of Liquid-crystalline Phase and those of gel Phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo Phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo Phase and investigated their physical properties using Phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo Phase in water were formed at 20−37 °C successfully, when these membranes contained ≥30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 °C, supporting that the membranes of these GUVs were in the lo Phase. To elucidate the interaction of a substance wi...