Docosahexaenoic acid (DHA) alters the structure and composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Membrane lipid microdomains are regions of the membrane thought to be functionally important, but which have remained poorly characterized because they have proven to be difficult to isolate. The exfoliation of small membranous vesicles from the cell surface is a continuous and normal activity in many cells. If microdomains are relatively large or stable, they may influence the structure and composition of exfoliated vesicles, which are easy to isolate. We tested the ability of docosahexaenoic acid (DHA), a fatty acid proposed to alter the structure of microdomains, to change the structure and composition of vesicles exfoliated from a murine leukemia cell line. Cells were cultured in normal and DHA-enriched media for 72 h, then washed and given a 15-h exfoliation period. Afterwards, the pooled vesicles and their parent plasma membrane were collected and analyzed. Vesicles and plasma membrane from cells grown in normal culture medium had similar fatty acid compositions, including equal, and low, proportions of DHA, but the vesicles had much more cholesterol and displayed higher anisotropy than the plasma membrane. When cells were grown in DHA-enriched medium, both the plasma membrane and exfoliated vesicles had 10-fold elevated levels of DHA in their phospholipids, with the DHA displacing other polyunsaturates. These cells released vesicles having significantly reduced levels of cholesterol and monoenoic fatty acids than those in normal culture. The anisotropy of these vesicles was also dramatically reduced. These data are consistent with DHA altering the structure and composition of membrane microdomains on the cell surface, and suggest that exfoliated vesicles may prove useful in the further study of membrane microdomains.

Comparison of phosphatidylcholines containing one or two docosahexaenoic acyl chains on properties of phospholipid monolayers and bilayers.

Docosahexaenoic acid (DHA) is the longest and most unsaturated of the n - 3 fatty acids found in membranes. Although a number of membrane properties have been demonstrated to be affected by the presence of this fatty acid, its mode of action has yet to be clearly elucidated. Prior reports on biological membranes have not distinguished the effect of mono-docosahexaenoyl phospholipids from those caused by phospholipids containing docosahexaenoic acid in both chains. This report compares properties of monolayers and bilayers composed of either 1-stearoyl-2-linolenoyl-sn-glycero-3-phosphocholine (as a control), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or 1,2-di-docosahexaenoyl-sn-glycero-3-phosphocholine. When compared to the mono-DHA phosphatidylcholine (PC), the di-DHA PC occupies a much larger area/molecule, supports a more fluid and permeable bilayer, and is less susceptible to peroxidation. Monolayers made from either phospholipid are not condensable by cholesterol. We suggest many of the membrane properties linked to the presence of DHA may be the result of phospholipids which have lost their normal positional selectivity and have incorporated DHA into both positions.

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers.

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.

Docosahexaenoic acid-containing phosphatidylcholine affects the binding of monoclonal antibodies to purified Kb reconstituted into liposomes.

Class I major histocompatibility complex (MHC I) molecules are transmembrane proteins that bind and present peptides to T-cell antigen receptors. The role of membrane lipids in controlling MHC I structure and function is not understood, although membrane lipid composition influences cell surface expression of MHC I. We reconstituted liposomes with purified MHC I (Kb) and probed the effect of lipid composition on MHC I structure (monoclonal anti-MHC I antibody binding). Four phospholipids were compared; each had a phosphocholine head group, stearic acid in the sn-1 position, and either oleic, alpha-linolenic, arachidonic, or docosahexaenoic acid (DHA) in the sn-2 position. The greatest binding of monoclonal antibody AF6-88.5, which detects a conformationally sensitive epitope in the extracellular region of the MHC I alpha-chain, was achieved with DHA-containing proteoliposomes. Other epitopes (CTKb, 5041.16.1) showed some sensitivity to lipid composition. The addition of beta2-microglobulin, which associates non-covalently with the alpha-chain and prevents alpha-chain aggregation, did not equalize antibody binding to proteoliposomes of different lipid composition, suggesting that free alpha-chain aggregation was not responsible for disparate antibody binding. Thus, DHA-containing membrane lipids may facilitate conformational change in the extracellular domains of the alpha-chain, thereby modulating MHC I function through effects on that protein's structure.

Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane.

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.

Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 microM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells.

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes.

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an (n-3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As (n-3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of (n-3) fatty acids from the diet, most likely due to retention of membrane-bound (n-3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1.

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose-response effect demonstrated that 5 or 10 microM DHA has no effect on Jurkat cell survival; however, 15 microM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 microM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.

Accessory molecules and antigen requirements for young and aging cytotoxic lymphocytes.

In this study I have investigated whether young and aging alloreactive cytotoxic T-lymphocytes (CTL) have comparable antigen requirements, and whether the accessory molecules Ly2 and LFA-1 participate similarly in cytolysis mediated by young and old cells. Spleen and lymph node cells from young and aging, naive and primed mice were stimulated in bulk culture with allogeneic cells; cytotoxicity and proliferation were then measured. Additionally, lymph node cells were cultured under limiting dilution conditions, and the resultant clones here tested for cytotoxicity in the presence or absence of antibodies against Ly2 and LFA-1. I found that accessory molecules from naive and primed, young and aging splenic CTL participated similarly in cytolysis. However, both cytotoxic and proliferating splenic T-cells from aging mice required a greater alloantigen concentration for optimal response. Heterogeneous lymph node CTL from old mice demonstrated less cytotoxicity than young CTL, and, on the clonal level, old naive CTL clones were less active than young clones. Naive aging clones were more resistant than young clones to anti-Ly2 and anti-LFA-1. After priming, clone activity among the various age groups did not differ. These results suggest a qualitative difference between young and old cytolytic cells prior to alloantigen priming in vivo.

Aged lymphocyte proliferation following incorporation and retention of dietary omega-3 fatty acids.

T cell activation involves events at the plasma membrane; therefore, molecules such as long chain omega-3 fatty acids that alter the structure of the plasma membrane may affect the activation of aged T cells. In this project we investigated whether the incorporation of omega-3 fatty acids (from fish oil), in the presence of vitamin E, improves age-diminished T cell proliferation. Young and old mice were fed diets rich in either fish (menhaden) oil or saturated fat for various lengths of time. Splenocytes were harvested from these mice and stimulated in culture with either mitogen or the antigen keyhole limpet hemocyanin (for a secondary response); proliferation was estimated by [3H]thymidine incorporation. We found no discernible effect of dietary omega-3 fatty acids (with vitamin E supplementation) on lymphocyte proliferation stimulated by the mitogens concanavalin A or phytohemagglutinin. We did, however, find that the saturated fat diet and the menhaden oil diet in young mice lowered protein kinase C activities in the particulate fractions of spleen cells when compared to chow-fed mice. Middle-aged and old mice were less affected by the experimental diets than young mice, but they demonstrated decreased protein kinase C activity as well. These alterations did not affect the ability of splenocytes to respond to mitogenic stimulation. Fatty acid analysis revealed that lymphocytes from mice fed saturated fat for 8.5 months retained significant amounts of the omega-3 fatty acid docosahexaenoic acid, despite the lack of dietary omega-3 fatty acids. However, when aged (but not young) lymphocytes were clonally expanded by antigen in vivo in the presence of dietary omega-3 fatty acids, they produced a greater secondary proliferative response than old lymphocytes expanded during a saturated fat diet. Although our results suggest that omega-3 fatty acids may enhance aged lymphocyte proliferation, the tenacious retention of these fatty acids makes comparison with omega-3-depleted lymphocytes difficult.

Kinetic assay of cytotoxic T lymphocyte clones. II. Comparison of primary and secondary responses and lectin-dependent cytotoxicity.

A kinetic assay for cytotoxic T lymphocyte (CTL) clones expanded briefly in vitro was used to compare CTL clones developing from the most frequent precursor population in naive spleens, among splenocytes primed in vitro, and among cells mediating lectin-dependent cellular cytotoxicity. Two kinetic parameters, K0 and n, were compared; K0 is the target cell concentration required for half-maximal velocity of killing, and n is the exponent of the target concentration in the Hill equation used for data analysis. The variance and frequency distribution of these parameters, rather than their numerical value, predicted differences in the clonal composition of distinct lymphoid populations assayed independently.

Kinetic assay of cytotoxic T lymphocyte clones. I. Methodology and data analysis.

A kinetic assay for cytotoxic T lymphocyte (CTL) clones expanded briefly in culture is described. The Hill equation for allosteric kinetics is used to fit curves to dose-response data. In this assay, K0--the target cell concentration required for half-maximal velocity of killing--is determined for clones generated from a population's most frequent CTL precursors. Because each clone's K0 is a constant under given conditions and a description of relative cytolytic efficiency, K0 values can be used to compare the clonal composition of disparate or dynamic populations. The parameter n is the target concentration exponent in the Hill equation and the slope of the line in the Hill plot. This parameter further specifies the shape of a clone's dose response curve, describing a clone's responsiveness to changes in target cell concentration. Because the assay is designed for small, undetermined effector cell concentrations, maximum velocities are not compared. The method described herein requires minimal supplies, equipment, and biological materials.

The use of urethane sponge matrix to assess in vivo recovery of murine cellular immunity following syngeneic bone marrow transplantation.

A unique method for assessing the in vivo recovery of cellular immunity following sublethal irradiation and syngeneic bone marrow transplantation of CBA/J mice is described. This method employs the serial surgical implantation of a urethane sponge matrix followed by impregnation of the sponge matrix with alloantigen. Sponge matrices were harvested from each mouse subject at 2 weeks, 4 weeks, and 8 weeks post transplant with and without treatment with interleukin-2. Recovery of cellular immunity was assessed as a function of cytotoxic T lymphocyte activity via cell-mediated lympholysis using chromium labeled targets. Repetitive procedures of implantation and removal of the matrices from the same animal were surgically feasible and were exceptionally well tolerated by the animals with insignificant morbidity thereby permitting continuous monitoring of immunologic recovery. Although the results of treatment with interleukin-2 were inconclusive, this method provides a sensitive, technically manageable means by which to assess an antigenic response in vivo at predetermined time intervals utilizing the same mouse subject. The method additionally affords the potential of being applicable to the in vivo assessment and manipulation of other aspects of the immune response.

Can docosahexaenoic acid inhibit metastasis by decreasing deformability of the tumor cell plasma membrane?

Murine leukemia cells were fused with small unilamellar vesicles composed of 1-stearoyl, 2-docosahexaenoylphosphatidylcholine. The docosahexaenoic acid (DHA)-modified cells were tested for deformability by forcing them through 5.0-microm Nucleopore filters. As the cellular DHA content increased, the cells passed through the filters with more difficulty. Furthermore, cells that passed through the filters had less DHA than cells that did not. Monitored by steady-state fluorescence polarization of membrane interior and surface probes, DHA reduced the membrane order in the hydrophobic interior while increasing the order at the aqueous interface. We attribute DHA's anti-metastatic properties in part to effects on membrane structure that reduce cell deformability.

Docosahexaenoic acid in phosphatidylcholine mediates cytotoxicity more effectively than other omega-3 and omega-6 fatty acids.

We reported previously that docosahexaenoic acid (22:6)-containing phosphatidylcholine (PC), but not oleic acid-containing PC nor 22:6-containing phosphatidylethanolamine, is toxic to tumor cells in vitro. To test whether other polyunsaturated fatty acids share 22:6's cytotoxic activity, we treated cultured T27A murine leukemia cells with PC liposomes composed of stearic acid in the sn-1 position and alpha-linolenic acid (alpha-18:3), arachidonic acid (20:4), or eicosapentaenoic acid (20:5) in the sn-2 position. PC containing 22:6 in both positions was also tested. Following treatment, the cells were monitored for fatty acid composition, liposome uptake and viability. Here we demonstrate that cytotoxicity is unique to 22:6-containing PCs and is not shared by PCs with other polyunsaturated omega-3 and omega-6 fatty acids. Because PCs with fatty acids other than 22:6 were taken up by cells but did not kill the cells, we propose that 22:6-containing PCs incorporated into cellular membranes produce unique changes in the membrane structure incompatible with cell survival. PC liposomes containing 22:6 are potential drug delivery vehicles that may, by virtue of their cytotoxicity, serve concomitantly as adjunct cancer therapy.

Immunologic, morphologic and chromosomal characterization of a cell line (TC78) established from a child with acute lymphoblastic leukemia.

We characterized a cell line established from bone marrow cells from a child with acute lymphoblastic leukemia. This cell line, TC78, had lymphoblastic morphology and was cytoplasmic peroxidase and esterase negative. The cells did not have T- or B-cell properties such as E- or EAC-rosette forming ability, reactivity with monoclonal T-cell or B2 antibodies, or immunoglobulin synthesis. We concluded that TC78 was a pre-pre B-cell line based on the following monoclonal antibody staining pattern: BA-1+, BA-2+, cALLa+, Ia+, 2H7+ and OKB2+. Growth in 'Dickie' culture and reactivity with 1G10 myeloid antibody suggested coexpression of lymphoid and myeloid characteristics. However, 1G10 expression proved dependent on culture conditions, illustrating one caveat in application of monoclonal antibodies in lineage determination.

Cholesterol versus cholesterol sulfate: effects on properties of phospholipid bilayers containing docosahexaenoic acid.

The important omega-3 fatty acid docosahexaenoic acid (DHA) is present at high concentration in some membranes that also contain the unusual sterol cholesterol sulfate (CS). The association between these lipids and their effect on membrane structure is presented here. Differential scanning calorimetry (DSC), MC540 fluorescence, erythritol permeability, pressure/area isotherms on lipid monolayers and molecular modeling are used to compare the effect of CS and cholesterol on model phospholipid membranes. By DSC, CS decreases the main phase transition temperature and broadens the transitions of dipalmitolyphosphatidylcholine (DPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1 PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6 PC) to a much larger extent than does cholesterol. In addition CS produces a three-component transition in 18:0,18:1 PC bilayers that is not seen with cholesterol. In a mixed phospholipid bilayer composed of 18:0,18:1 PC/18:0,22:6 PC (1:1, mol/mol), CS at 2.5 membrane mol% or more induces lateral phase separation while cholesterol does not. CS decreases lipid packing density and increases permeability of 18:0,18:1 PC and 18:0,22:6 PC bilayers to a much larger extent than cholesterol. CS disrupts oleic acid-containing bilayers more than those containing DHA. Molecular modeling confirms that the anionic sulfate moiety on CS renders this sterol more polar than cholesterol with the consequence that CS likely resides higher (extends further into the aqueous environment) in the bilayer. CS can therefore be preferentially accommodated into DHA-enriched bilayers where its tetracyclic ring system may fit into the delta 4 pocket of DHA, a location excluded to cholesterol. It is proposed that CS may in part replace the membrane function of cholesterol in DHA-rich membranes.

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes.

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.