Surface shape change during fusion of erythrocyte membranes is sensitive to membrane skeleton agents.

We previously reported that the induction of membrane fusion between pairs of erythrocyte ghosts is accompanied by the formation of a multipore fusion zone that undergoes an area expansion with condition-dependent characteristics. These characteristics allowed us to hypothesize substantial, if not major, involvement of the spectrin-based membrane skeleton in controlling this expansion. It was also found that the fusion zone, which first appears in phase optics as a flat diaphragm, has a lifetime that is also highly condition-dependent. We report here that 2,3-diphosphoglycerate, wheat germ agglutinin, diamide, and N-ethylmaleimide, all known to have binding sites primarily on skeleton components (including spectrin), have condition-dependent effects on specific components of the fusion zone diameter versus time expansion curve and the flat diaphragm lifetime. We also report a pH/ionic strength condition that causes a dramatic stabilization of flat diaphragms in a manner consistent with the known pH/ionic strength dependence of the spectrin calorimetric transition, thus further supporting the hypothesis of spectrin involvement. Our data suggest that the influence of the membrane skeleton on cell fusion is to restrain the rounding up that takes place after membrane fusion and that it may have variable, rather than fixed, mechanical properties. Data show that WGA, a known ligand for sialic acid, and DPG, a known metabolite, influences the flat diaphragm stability and late period expansion rates, raising the possibility that some of these mechanical properties are biologically regulated.     

Membrane skeleton involvement in cell fusion kinetics: a parameter that correlates with erythrocyte osmotic fragility.

Spectrin levels in erythrocytes have been related to several biomechanical and biophysical membrane properties essential to the survival and function of the cell. Populations of erythrocytes display a natural and finite range of sensitivities to osmotic shock that has been directly correlated, in studies from other laboratories, to the presence of spectrin. We used a procedure to isolate subpopulations of 1) the osmotically most sensitive and 2) the osmotically most resistant erythrocyte membranes in an attempt to select for membranes enriched and depleted in spectrin (and/or a related component). The mechanical function of the spectrin-based membrane skeleton was further explored in these two subpopulations by searching for any effect on the time-dependent increase in fusion zone diameter in pairs of electrofused erythrocyte ghosts as a model for cell fusion. The results clearly show that the diameter expansions in fusions of membranes from osmotically resistant erythrocytes are faster in the early stage (up to 9 to 10 s after fusion) but do not thereafter expand as far as in fusions of membranes from osmotically sensitive membranes.     

Membrane labelling by fluorescent probes: incorporation of TMA-DPH in erythrocyte membranes.

A study of the labelling of isolated resealed erythrocyte membranes by TMA-DPH has been carried out. A quantitative study shows that saturation appears to take place when increasing the relative quantity of probe bulk concentration to membrane concentration; this is readily interpreted by a simple incorporation model with a limited number of sites in the membrane. A qualitative study shows that an increase in the labelling leads to an evolution of the probe fluorescence properties; the existence of different types of sites is involved in the interpretation but the system is too complex to allow it to be represented by a simple model. As a consequence of this study, care has to be taken in labelling biological material so as to avoid excessive probe incorporation.     

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, and alpha-L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of alpha-D-glucosidase to 70% of beta-D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of beta-D-galactosidase to 89% of alpha-D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of beta-D-galactosidase to 85% of alpha-D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.     

HVJ-induced fusion of liposome to erythrocyte. Possible involvement of virus-induced activated state of erythrocyte membranes during fusion process

HVJ-induced fusion between liposomes and erythrocytes was examined as a model of cell-cell fusion and the following results were obtained: Liposome-liposome fusion seldom occurred in the presence of HVJ. Liposomes free of receptors for viral HN protein could efficiently fuse with erythrocytes when the concentration of liposomes and erythrocytes was high. Direct interaction between HVJ and liposomes should be negligible in the present system. A distinct lag phase (10 min) was observed before the liposome-erythrocyte fusion occurred appreciably. Efficiency of the liposome-erythrocyte fusion decreased linearly up to 1 h after stopping the virus-erythrocyte fusion. The former fusion was almost negligible after 1 h but could be restored by further addition of HVJ. Efficiency of the liposome-erythrocyte fusion was suppressed significantly when erythrocytes were pretreated with N-ethylmaleimide (N-EM), a membrane-permeable sulfhydryl agent, whereas rho-chloromercuriphenylsulfonic acid (PCMBS), a membrane-impermeable sulfhydryl agent, had no appreciable effect on the fusion efficiency. The study suggests possible involvement of some HVJ-induced 'activated state' of the erythrocyte membrane during the process inducing the liposome-erythrocyte fusion. The structure including the membrane skeleton system may be responsible for producing an 'activated state' in the erythrocyte membrane. Such an 'activated state' was induced 10 min after the viral infection to erythrocytes, and thereafter decayed gradually.     

Relationship of major phosphorylation reactions and MgATPase activities to ATP-dependent shape change of human erythrocyte membranes

Human erythrocyte ghosts prepared by hemolysis and washing in hypotonic Tris are crenated by salt and divalent cations, but undergo shape change to smooth biconcave discs and stomatocytic forms when incubated with MgATP at 37 degrees C. This is normally accompanied by protein and lipid phosphorylations in which the major phosphate acceptors are the spectrin beta-chain and inositol phospholipids, respectively. The system was manipulated in several ways to demonstrate the independence of ATP-dependent shape change from the major phosphorylation reactions. Salt-extracted membranes incubated with adenosine, an inhibitor of spectrin and phosphatidylinositol kinases, underwent normal shape change despite reductions of greater than 90% in spectrin and phospholipid labeling by [gamma-32P]ATP. ATP-dependent shape change was blocked by vanadate at micromolar concentrations (half-maximal inhibition at less than 1 microM), but vanadate did not inhibit membrane autophosphorylation reactions or turnover of spectrin- or lipid-bound phosphate. Vanadate inhibited part of the ATP hydrolysis that accompanies shape change and is expressed in the presence of ouabain and EGTA. The vanadate-sensitive MgATPase activity was approximately 3 nmol Pi X min-1 X mg of protein-1. The results implicate it in ATP-dependent shape change.     

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells.

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degree C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipids.     

Fusion between Sendai virus envelopes and biological membranes. The use of fluorescent probes for quantitative estimation of virus-membrane fusion

The fluorescent probes, N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine and lissamine-rhodamine-B-sulfonylphosphatidylethanolamine, were inserted at the appropriate surface density into membranes of reconstituted Sendai virus envelopes, thus allowing transfer of energy between the fluorescent probes. In addition, only the fluorescent molecule N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine was inserted into the viral envelopes, resulting in self-quenching. Incubation of fluorescent, reconstituted Sendai virus envelopes with human erythrocyte ghosts resulted in either reduction in the efficiency of energy transfer or in fluorescence dequenching. No reduction in the efficiency of energy transfer or fluorescence dequenching was observed when fluorescent, reconstituted Sendai virus envelopes were incubated with glutaraldehyde-fixed or desialized human erythrocyte ghosts. Similarly, no change in the fluorescence value was observed when nonfusogenic, reconstituted Sendai virus envelopes were incubated with human erythrocyte ghosts. These results clearly show that reduction in the efficiency of energy transfer or dequenching is due to virus-membrane fusion and not to lipid-lipid exchange. Incubation of reconstituted Sendai virus envelopes, carrying inserted N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine, with cultured cells also resulted in a significant and measurable dequenching. However, incubation of nonfusogenic, fluorescent reconstituted Sendai virus envelopes with hepatoma tissue culture cells also resulted in fluorescent dequenching, the degree of which was about 50% of that observed with fusogenic, fluorescent reconstituted viral envelopes. It is therefore possible that, in addition to virus-membrane fusion, endocytosis of fluorescent viral envelopes results in fluorescence dequenching as well.     

Change in electrophoretic mobility of human erythrocyte as the result of membrane shape change in vitro.

Shape change of human erythrocyte due to the membrane externalization induced by incorporation of excess lysolecithin or by hypertonic treatment in vitro, was accompanied by a decrease in the electrophoretic mobility of the cell. No change in sialic acid content of the membrane, mostly responsible for the cell surface charge, was observed in any of these treatments. Therefore, the membrane externalization seems to cause a redistribution of the surface charge and the resulting alteration of the electrophoretic mobility of the cell. Heating at 48 degrees C for 1 hour induced both the shape change and change in the mobility, but at the same time it reduced the membrane sialic acid content.     

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degrees C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipds.     

The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes

The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.     

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes.

Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.     

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.     

Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein.

We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars.     

Chicken erythrocyte membranes: Comparison of nuclear and plasma membranes from adults and embryos

Nuclear membranes and plasma membranes of chicken erythrocytes were tested for some enzyme activities and polypeptide content. Both membranes show ATPase and NADH cytochrome c reductase activities, which were higher for nuclear membrane preparations; besides the nuclear membrane ATPase is not stimulated by Na + K + ions. SDS solubilization of these membranes followed by separation using SDS polyacrylamide gel electrophoresis yields about 25 bands. Nuclear membranes and plasma membranes possess specific bands; some differences were seen when adult and embryonic membranes were compared.     

Membrane fusion by single influenza hemagglutinin trimers. Kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles

Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis. The surface density of hemagglutinin fusion-activity sites on the vesicles was varied, while keeping the surface density of receptor-binding activity sites constant, by co-reconstitution of the fusogenic form of hemagglutinin, HA(1,2), and the non-fusogenic form, HA(0), at various HA(1,2):(HA(1,2) + HA(0)) ratios. The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy. The best fit of a log-log plot of fusion rate versus the surface density of HA(1,2) exhibited a slope of 0.85, strongly supporting the hypothesis that single hemagglutinin trimers are sufficient for fusion. When only HA(1,2) (without HA(0)) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA(1,2) was distinct from that for the HA(1,2)-HA(0) co-reconstitution. The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers.     

Chicken erythrocyte membranes: comparison of nuclear and plasma membranes from adults and embryos

These experiments were designed to determine whether the migration of RNA molecules from an implanted nucleus to the host cytoplasm and from there into the host cell nucleus against a concentration gradient might reflect an artefact induced by the process of nuclear transplantation. That is, are RNA molecules, as previously shown for certain nuclear proteins, caused to artefactually leave a manipulated nucleus and then move into the host cell nucleus (as well as return to the grafted nucleus) during the recovery period?A variety of experiments involving different kinds of manipulative sequences and different numbers of nuclear transplantations suggest—but do not prove—that no artefact is involved in the migration of RNA from one nucleus to another but two experiments strongly support the view that the shuttling activity is a normal physiological process. One of the latter involved a determination of the rate of egress of ³H-RNA from an implanted nucleus and reveals that that rate, in contrast with the equivalent rate of egress for labeled proteins which is clearly abnormal after micromanipulation, is totally consonant with the rate of movement of RNA from nucleus to cytoplasm established from experiments that do not involve micromanipulation. The other experiment involves comparison of (1) the amount of radioactivity acquired by an unlabeled nucleus present in the cell at the time a labeled nucleus is implanted with (2) the amount of radioactivity acquired by an unlabeled nucleus implanted after a labeled nucleus had been implanted and had time to recover from any possible operation-induced trauma. With ³H-protein nuclei the host nuclei of (1) acquired much more label than the host nuclei of (2) because in (1) the host nuclei were able to acquire much of the artefactually-released ³H-protein. For the ³H-RNA experiments, however, little difference was found between (1) and (2) in the amount of label acquired by the host cell nuclei. It can be concluded that little, if any, of the non-random shuttling activity of RNA molecules can be a reflection of an artefact.     

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes.

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparation. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium d?odosalicylate are recoverd in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.