Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (~ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.     

The pH-dependent distribution of the photosensitizer chlorin e6 among plasma proteins and membranes: a physico-chemical approach

Decrease in interstitial pH of the tumor stroma and over-expression of low density lipoprotein (LDL) receptors by several types of neoplastic cells have been suggested to be important determinants of selective retention of photosensitizers by proliferative tissues. The interactions of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic groups, with plasma proteins and DOPC unilamellar vesicles are investigated by fluorescence spectroscopy. The binding constant to liposomes, with reference to the DOPC concentration, is 6 x 10(3) M(-1) at pH 7.4. Binding of Ce6 to LDL involves about ten high affinity sites close to the apoprotein and some solubilization in the lipid compartment. The overall association constant is 5.7 x 10(7) M(-1) at pH 7.4. Human serum albumin (HSA) is the major carrier (association constant 1.8 x 10(8) M(-1) at pH 7.4). Whereas the affinity of Ce6 for LDL and liposomes increases at lower pH, it decreases for albumin. Between pH 7.4 and 6.5, the relative affinities of Ce6 for LDL versus HSA, and for membranes versus HSA, are multiplied by 4.6 and 3.5, respectively. These effects are likely driven by the ionization equilibria of the photosensitizer carboxylic chains. Then, the cellular uptake of chlorin e6 may be facilitated by its pH-mediated redistribution within the tumor stroma.     

The pH-dependent distribution of the photosensitizer chlorin e6 among plasma proteins and membranes: A physico-chemical approach

Decrease in interstitial pH of the tumor stroma and over-expression of low density lipoprotein (LDL) receptors by several types of neoplastic cells have been suggested to be important determinants of selective retention of photosensitizers by proliferative tissues. The interactions of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic groups, with plasma proteins and DOPC unilamellar vesicles are investigated by fluorescence spectroscopy. The binding constant to liposomes, with reference to the DOPC concentration, is 6 × 10³ M^− 1 at pH 7.4. Binding of Ce6 to LDL involves about ten high affinity sites close to the apoprotein and some solubilization in the lipid compartment. The overall association constant is 5.7 × 10⁷ M^− 1 at pH 7.4. Human serum albumin (HSA) is the major carrier (association constant 1.8 × 10⁸ M^− 1 at pH 7.4). Whereas the affinity of Ce6 for LDL and liposomes increases at lower pH, it decreases for albumin. Between pH 7.4 and 6.5, the relative affinities of Ce6 for LDL versus HSA, and for membranes versus HSA, are multiplied by 4.6 and 3.5, respectively. These effects are likely driven by the ionization equilibria of the photosensitizer carboxylic chains. Then, the cellular uptake of chlorin e6 may be facilitated by its pH-mediated redistribution within the tumor stroma.     

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Interactions of apomyoglobin with membranes: mechanisms and effects on heme uptake

The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged phospholipids. The pH dependence of apomyoglobin interactions with membranes is a two-step process, and involves a partially folded state stabilized at acidic pH. An evident role for the interaction of apomyoglobin with lipid bilayers would be to facilitate the uptake of heme from the outer mitochondrial membrane. However, heme binding to apomyoglobin is observed at neutral pH when the protein remains in solution, and slows down as the pH becomes more favorable to membrane interactions. The effective incorporation of soluble heme into apomyoglobin at neutral pH suggests that the interaction of apomyoglobin with membranes is not necessary for the heme uptake from the lipid bilayer. In vivo, however, the ability of apomyoglobin to interact with membrane may facilitate its localization in the vicinity of the mitochondrial membranes, and so may increase the yield of heme uptake. Moreover, the behavior of apomyoglobin in the presence of membranes shows striking similarities with that of other proteins with a globin fold. This suggests that the globin fold is well adapted for soluble proteins whose functions require interactions with membranes.     

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Oxygen distribution in the fluid/gel phases of lipid membranes

The interactions between oxygen and lipid membranes play fundamental roles in basic biological processes (e.g., cellular respiration). Obviously, membrane oxidation is expected to be critically dependent on the distribution and concentration of oxygen in the membrane. Here, we combined theoretical and experimental methods to investigate oxygen partition and distribution in lipid membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a temperature range between 298 and 323 K, specifically focusing on the changes caused by the lipid phase and phase transition. Even though oxygen is known to be more concentrated in the center of fluid phase membranes than on the headgroup regions, the distribution profile of oxygen inside gel-phase bilayers remained to be determined. Molecular dynamics simulations now show that the distribution of oxygen inside DPPC bilayers dramatically changes upon crossing the main transition temperature, with oxygen being nearly depleted halfway from the headgroups to the membrane center below the transition temperature. In a parallel approach, singlet oxygen luminescence emission measurements employing the photosensitizer Pheophorbide-a (Pheo) confirmed the differences in oxygen distribution and concentration profiles between gel- and fluid-phase membranes, revealing changes in the microenvironment of the embedded photosensitizer. Our results also reveal that excited triplet state lifetime, as it can be determined from the singlet oxygen luminescence kinetics, is a useful probe to assess oxygen distribution in lipid membranes with distinct lipid compositions.     

Morphological study on the entry of African swine fever virus into cells

The early interactions between African swine fever virus (ASFV) and monkey kidney cells in culture, and the effect of chloroquine were studied by electron microscopy. Our results indicate that ASFV uptake occurs by endocytosis: after attachment to the cell surface, the virions were seen in coated pits and were internalized by endocytosis in endosomes and finally in lysosomes. Virions in coated vesicles were never seen. All these steps were completed in about 15 min. Direct penetration of viruses through the plasma membrane was never observed. In order to elucidate the participation of an acidic intracellular compartment in the penetration of ASFV, we studied the effect of chloroquine, a lysosomotropic drug known to increase the pH of acidic intracellular vacuoles and to inhibit ASFV infection. In the presence of this drug there were no apparent alterations on binding, endocytosis and intracellular distribution of the viral particles. The main effect of chloroquine was to retain the virions in lysosomes. When the drug was removed from the medium, the viral particles disappeared and images of binding of viral membranes with the membranes of the intracellular vacuoles were obtained, suggesting that the inhibited step is the uncoating of the virus. Viral fusion with the plasma membrane was obtained when the medium was acidified to pH 5-6. These results suggest that ASFV enters the cells by adsorptive endocytosis and that the uncoating process takes place intracellularly in a way similar to that described for Semliki Forest virus and other enveloped viruses.     

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1–positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of “Trojan horse” antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Pharmacological inhibition of the intracellular transport of low-density lipoprotein-derived cholesterol in Chinese hamster ovary cells

Mammalian cells, cultured in the presence of serum lipoproteins, acquire cholesterol necessary for growth from the uptake and lysosomal hydrolysis of low-density lipoproteins (LDL). The mechanism(s) of intracellular transport of LDL-derived cholesterol from lysosomes to other cellular sites is unknown. In this study, various pharmacological agents were assessed for their ability to inhibit the movement of LDL-cholesterol from lysosomes to the plasma membrane. The only pharmacological agent tested in these experiments that specifically inhibited LDL-cholesterol movement was U18666A. Ketoconazole impaired the intracellular transport of LDL-cholesterol; however, ketoconazole also had a general effect on cholesterol movement, since it impeded the desorption of endogenously synthesized cholesterol into the medium. Other drugs that affected cholesterol movement appeared to be nonspecific. Cholesterol transport from lysosomes to plasma membranes was not significantly altered by agents that affect lysosomal function or cytoskeletal organization, as well as energy poisons and cycloheximide.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Hydrolase secretion is a consequence of membrane recycling

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.     

Uptake of gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein by cultured rat granulosa cells: cellular mechanisms involved in lipoprotein metabolism and their importance to steroidogenesis

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold-LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34-38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold-LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold-labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL-cholesterol vital for steroidogenesis.     

Itinerary of high density lipoproteins in endothelial cells

High density lipoprotein (HDL) and its main protein component apolipoprotein A-I (ApoA-I) have multiple anti-atherogenic functions. Some of them are exerted within the vessel wall, so that HDL needs to pass the endothelial barrier. To elucidate their itinerary through endothelial cells (ECs), we labelled ApoA-I and HDL either fluorescently or with 1.4 nm nanogold and investigated their cellular localization by using immunofluorescent microscopy (IFM) and electron microscopy (EM). HDL as well as ApoA-I is taken up by ECs into the same route of intracellular trafficking. Time kinetics and pulse chase experiments revealed that HDL is trafficked through different vesicles. HDL partially co-localized with LDL, albumin, and transferrin. HDL did not co-localize with clathrin and caveolin-1. Fluorescent HDL was recovered at small proportions in early endosomes and endosome to trans-golgi network vesicles but not at all in recycling endosomes, in late endosomes or lysosomes. EM identified HDL mainly in large filled vesicles which however upon IFM did not colocalize with markers of multivesicular bodies or autophagosomes. The uptake or cellular distribution of HDL was altered upon pharmacological interference with cytochalasine D, colchicine and dynasore. Blockage of fluid phase uptake with Amiloride or EIPA did not reduce the uptake of HDL. Neither did we observe any co-localization of HDL with dextran as the marker of fluid phase uptake. In conclusion, HDL and ApoA-I are internalized and trafficked by endothelial cells through a non-classical endocytic route.     

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids. LDL internalization is not required for cellular lipid uptake. Collectively, our data provide evidence that oxidized phospholipids, owing to their high exchangeability between lipoproteins and cell membranes, may act within a short time on different cellular sites in VSMCs and affect various lipid and protein components through physical or chemical interactions, which might then serve as starting points for intracellular signaling.     

Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts

Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-β-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed.     

Self-assembled vesicles of monocarboxylic acids and alcohols: conditions for stability and for the encapsulation of biopolymers

We tested the ability of saturated n-monocarboxylic acids ranging from eight to 12 carbons in length to self-assemble into vesicles, and determined the minimal concentrations and chain lengths necessary to form stable bilayer membranes. Under defined conditions of pH and concentrations exceeding 150 mM, an unbranched monocarboxylic acid as short as eight carbons in length (n-octanoic acid) assembled into vesicular structures. Nonanoic acid (85 mM) formed stable vesicles at pH 7.0, the pK of the acid in bilayers, and was chosen for further testing. At pH 6 and below, the vesicles were unstable and the acid was present as droplets. At pH ranges of 8 and above clear solutions of micelles formed. However, addition of small amounts of an alcohol (nonanol) markedly stabilized the bilayers, and vesicles were present at significantly lower concentrations (∼20 mM) at pH ranges up to 11. The formation of vesicles near the pK_a of the acids can be explained by the formation of stable RCOO⁻…HOOCR hydrogen bond networks in the presence of both ionized and neutral acid functions. Similarly, the effects of alcohols at high pH suggests the formation of stable RCOO⁻…HOR hydrogen bond networks when neutral RCOOH groups are absent. The vesicles provided a selective permeability barrier, as indicated by osmotic activity and ionic dye capture, and could encapsulate macromolecules such as DNA and a protein. When catalase was encapsulated in vesicles of decanoic acid and decanol, the enzyme was protected from degradation by protease, and could act as a catalyst for its substrate, hydrogen peroxide, which readily diffused across the membrane. We conclude that membranous vesicles produced by mixed short chain monocarboxylic acids and alcohols are useful models for testing the limits of stabilizing hydrophobic effects in membranes and for prebiotic membrane formation.