Effect of lipid compositions on gene transfer into 293 cells using Sendai F/HN-virosomes

Fusogenic liposomes that incorporate Sendai virus envelope proteins, so-called Sendai virosomes, have been developed for in vitro and in vivo genetic modification of animal cells. In this study, several different virosomes of varying lipid compositions were formulated and their in vitro gene-transfer efficiencies compared. The virosomes were prepared by quantitative reconstitution of the Sendai envelope, fusion (F) and hemagglutinin-neuraminidase (HN) proteins into liposomal vesicles. Virosomes that contained luciferase reporter genes were tested in 293 transformed human kidney cells. F/HN-virosomes that were prepared with an artificial Sendai viral envelope (ASVE-virosomes) or phosphatidylserine (PS-virosomes) exhibited an 8- or 6-fold higher gene-transfer efficiency than cationic liposomes that were made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). F/HNvirosomes that were prepared with phosphatidic acid (PA-virosomes) instead of PS were less efficient in gene transfer than either ASVE- or PS-virosomes. In addition, the gene-transfer capability of ASVE- and PS-virosomes was maximal at a Ca2+ concentration of 510 mM. These results suggest that the incorporated lipid components significantly affect the in vitro gene transfer that is mediated by Sendai F/HN-virosomes.     

Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.     

Reconstitution of fusogenic Sendai virus envelopes by the use of the detergent chaps

Sendai virus envelopes have been a useful tool in studying the mechanism of membrane-membrane fusion and have served as a vehicle for introducing foreign molecules (e.g., membrane proteins) into recipient cells. Reconstituted Sendai virus envelopes are routinely obtained following solubilization of virus particles with Triton X-100. This detergent has a low critical micellar concentration which precludes it from being the best detergent of choice in reconstitution studies. Nevertheless, it has remained in use since other detergents such as sodium deoxycholate and sodium cholate rendered the resultant vesicles inactive. Triton X-100 may be suboptimal for studies of some proteins that need be coreconstituted with the viral envelopes. Thus, alternative advantageous detergents, which retain the envelope fusogenic activity, have been sought. In this study we show that the synthetic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) effectively solubilizes the Sendai virions, and that the vesicles formed by simple reconstitution protocols appear structurally and biochemically similar to those obtained with Triton X-100. The resultant vesicles retain functional integrity as assessed in both fusion and hemolysis assays. This protocol seems to be useful in sendai envelope-mediated reimplantation of Fc epsilon receptors into the plasma membranes of rat basophilic leukemia cells.     

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ～37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.     

Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin

A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze-fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

Rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

Fertilization in the sea urchin is accompanied by rapid reorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of membranes used in assembling the male pronuclear envelope in vitro. We provide here biochemical evidence for the rearrangement of sea urchin egg cytoplasmic membrane domains at fertilization up to the first mitosis, with respect to two nuclear envelope markers, lamin B and lamin B receptor (LBR), using purified vesicles prepared from homogenates fractionated by floatation on sucrose gradients. In unfertilized eggs, immunoprecipitation data indicate that most of lamin B and LBR are localized in the same vesicles but do not interact. By 3 min post-fertilization, both proteins are more widely distributed across the gradients and by 12 min most of lamin B and LBR are localized in vesicles of different densities. This partitioning is maintained throughout S phase. At mitosis, most lamin B and LBR remain in distinct vesicles, while a small proportion of lamin B and LBR, likely derived from the disassembled nuclear envelope, associate in a minor subset of vesicles. The results illustrate a dynamic reorganization of egg cytoplasmic membranes at fertilization, and the establishment of distinct membrane domains enriched in specific nuclear envelope markers during the first cell cycle of sea urchin development. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosol and membranes originate from fertilized but not unfertilized eggs, suggesting that fertilization-induced membrane rearrangements contribute to the ability of the egg to assemble the male pronuclear envelope.     

Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.     

Outer membrane of Salmonella typhimurium: Reconstitution of sucrose-permeable membrane vesicles

Outer membrane vesicles were reconstituted from phospholipids, lipopolysaccharide, and outer membrane proteins isolated from Salmonella typhimurium. The vesicles appeared to be permeable to sucrose and other small oligosaccharides only when membrane proteins were added to the reconstitution system. The size of saccharides that could pass through the vesicle membranes was found to be close to the size of saccharides that penetrate through the intact outer membrane of S. typhimurium.     

Localization of enzymes in specialized regions of the microsomal membrane

Microsomal vesicle were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADPH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patchers in the microsomal membrane.     

Purification and characterization of a cortical secretory vesicle membrane fraction

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.     

Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles

Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth. Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells. Enterotoxigenic E. coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation. Vesicle protein profiles were similar but not identical to outer membranes and differed between strains. Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal. Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles. Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles. In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains.     

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.     

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.     

Isolation and structure of the lipid envelopes from the nitrogen-fixing vesicles of Frankia sp. strain CpI1.

Frankia vesicles are differentiated during nitrogen starvation; they contain nitrogenase whether produced by free-living frankiae or by frankiae in actinorhizal root nodules. Vesicles are surrounded by envelopes of several monolayers of uncharacterized lipid. It has been suggested that the envelope limits diffusion of O2 into the vesicle cytoplasm, thereby preventing inactivation of nitrogenase. Whole vesicles were prepared on sucrose gradients and sonicated, and vesicle envelopes were isolated on top of a cushion of 40% sucrose. Transmission electron microscopy of potassium permanganate-fixed envelopes confirmed the purity of these preparations. Only the outer and inner envelope layers were visible in permanganate-fixed intact vesicles; the laminae were not visible in aldehyde-osmium-fixed, lead citrate-uranyl acetate-stained whole vesicles. However, the laminated nature of the envelope was clearly evident in sonicated vesicles and in envelope fragments fixed with KMnO4. The observations indicate that partial disruption of the vesicle envelope enables its visualization with permanganate fixation, and these observations open the way for further studies on the relationship of the vesicle surface to environmental conditions.