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Studies of membrane fusion. IV. Fusion of HeLa cells with Sendai virus.

The Sendai virus-induced fusion of HeLa cells has been studied by freeze-fracture electron microscopy. Freeze-fracture observations confirm previous scanning electron-microscope studies (1977) and show that at 4 degrees C virus particles bind to the cell surface and that cell agglutination results from the crosslinking by virus particles of microvilli on adjacent cells. Incubation at 37 degrees C initiates a change in viral envelope structure and fusion of 'altered' virus particles with the cell plasma membrane. Fusion of a virus particle with two crosslinked cells is probably the membrane fusion event which initiates cell-cell fusion; fusion is completed as a result of virally induced cell swelling. Lateral diffusion of viral envelope components following virus-cell fusion and, in some instances, an aggregation of plasma membrane intramembrane particles occurs in swollen cells. These observations show that the mechanisms of viral envelope-cell and probably cell-cell fusion are the same as have been reported for erythrocytes. Although endocytosis of intact virus particles does occur, the specialized cell-mediated mechanism for fusion of the viral envelope with the cell plasma membrane suggests that this, and not viropexis, is the mechanism of Sendai virus infection.

Agglutination

In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

Amoeba

Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus.

The fusion of human erythrocytes with non-haemolytic '1-day' Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic '3-day' Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic '3-day' Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.

Cell Fusion

Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals.

The effect of divalent metals on the interaction and mixing of membrane components in vesicles prepared from acidic phospholipids has been examined using freeze-fracture electron microscopy and differential scanning calorimetry. Ca2+, and to a certain extent Mg2+, induce extensive mixing of vesicle membrane components and drastic structural rearrangements to form new membranous structures. In contrast to the mixing of vesicle membrane components in the absence of Ca2+ described in the accompanying paper which occurs via diffusion of lipid molecules between vesicles, mixing of membrane components induced by Ca2+ or Mg2+ results from true fusion of entire vesicles. There appears to be a "threshold" concentration at which Ca2+ and Mg2+ become effective in inducing vesicle fusion and the threshold concentration varies for different acidic phospholipid species. Different phospholipids also vary markedly in their relative responsiveness to Ca2+ and Mg2+, with certain phospholipids being much more susceptible to fusion by Ca2+ than Mg2+. Vesicle fusion induced by divalent cations also requires that the lipids of the interacting membranes be in a "fluid" state (T greater than Tc). Fusion of vesicle membranes by Ca2+ and Mg2+ does not appear to be due to simple electrostatic charge neutralization. Rather the action of these cations in inducing fusion is related to their ability to induce isothermal phase transitions and phase separations in phospholipid membranes. It is suggested that under these conditions membranes become transiently susceptible to fusion as a result of changes in molecular packing and creation of new phase boundaries induced by Ca2+ (or Mg2+).

Calcium

XC cell fusion by murine leukemia viruses: fusion from without.

Concentrated murine leukemia virus (MuLV) or MuLV producing cells induce XC cell fusion within an hour leading to syncytia formation. While MuLV inactivated by UV irradiation, beta-propiolactone or hydroxylamine treatment still caused cell fusion, Bromelin- or trypsin treated MuLV was no longer able to fuse XC cells. Though sonicated MuLV induced no XC cell fusion, it interfered with cell fusion as caused by untreated MuLV. XC cells infected by diluted MuLV of a titer lower than 1 X 10(5) PFU/ml formed no syncytia although they produced MuLV. The cell fusion mechanism is discussed.

Animals

Temperature dependence of sperm-egg fusion and post-fusion events in hamster fertilization.

The effects of temperature (4--37 degrees C) on sperm-egg fusion and the post-fusion events were studied. At 4--10 degrees C, acrosome-reacted spermatozoa bound to egg plasma membranes, but could not fuse with them. At 25 degrees C or above, both binding and fusion took place. The post-fusion events could occur over a broad temperature range (4--37 degrees C) but the events progressed faster with increasing temperature. An abnormal development of egg pronucleus, possibly due to an incomplete functioning of the meiotic spindle mechanism, was observed in eggs inseminated at 37 degrees C and cultured at 25 degrees C.

Animals

Studies of membrane fusion. I. Paramyxovirus-induced cell fusion, a scanning electron-microscope study.

Fusion of erythrocytes and HeLa cells with Sendai and Newcastle disease viruses has been studied by scanning electron microscopy. Most virus particles are spherical but vary in diameter from approximately 200 to approximately 600 nm. At 4 degrees C virus particles bind randomly to the cell surface and at high cell densities cross-linking of adjacent cells by virus particles results in cell agglutination. Cell-cell fusion takes place when the agglutinated cell suspension is warmed to 37 degrees C. Fusion is initiated at sites of cell-cell contact and is accompanied in all cases by cell swelling. In the case of suspension HeLa cells, virally mediated cell swelling involves an 'unfolding' of cell surface microvilli and results in the formation of smooth-surfaced single or fused cells. With erythrocytes, swelling results in haemolysis. There is a dramatic reduction in the numbers of virus particles bound to cells following fusion.

Cell Aggregation

[Kinetics and ultrastructure of sheep fibroblast fusion induced by polyethylene glycol. Comparison with endogenous cell fusion induced by Visna virus].

The authors compare the fusion of sheep fibroblasts induced by low multiplicities of infection using visna virus and by high concentrations of polyethylene-glycol. In the case of Visna virus cell fusion is of the endogenous type, while fusion induced by polyethylene-glycol is of the exogenous type. The ultrastructural features are discussed for each type of cell fusion. The main differences between the two systems involve the intracellular microfilaments and Golgi apparatus.

Animals

Oblique Lateral Interbody Fusion With Lateral Vertebral Screw Fixation Versus Transforaminal Lumbar Interbody Fusion for Severe Lumbar Stenosis: Results of a Multicenter Randomized Controlled Trial.

BACKGROUND AND OBJECTIVES: The benefits of oblique lateral interbody fusion (OLIF) vs transforaminal lumbar interbody fusion (TLIF) in severe lumbar stenosis (Schizas C/D) remain uncertain. This randomized trial compared clinical, radiographic, and safety outcomes of OLIF and TLIF. METHODS: From November 2018 to December 2021, a prospective, multicenter, randomized controlled trial enrolled 260 adults with single-level severe stenosis and instability. In total, 224 patients were randomized to OLIF or TLIF. Prespecified outcomes followed consolidated standards of reporting trials. Primary outcomes were visual analog scale back/leg pain and Oswestry Disability Index (ODI), with minimal clinically important difference thresholds of ODI &#x2265;12-13 points or &#x2265;30% improvement, and visual analog scale &#x2265;1.5-2.0 points. Radiographic measures included disc height, lumbar and segmental lordosis, and canal cross-sectional area (CSA). Complications were recorded. Ethics approval was obtained from the institutional review board, the trial was registered with ISRCTN.com , and all patients provided written informed consent. RESULTS: In total, 224 patients were randomized, 5 were lost to follow-up (TLIF n = 2, OLIF n = 3). Baseline features were comparable. OLIF was associated with shorter operative time, less blood loss, earlier ambulation, and shorter hospital stay (all P < .05). Both groups achieved significant, clinically meaningful improvements. OLIF showed greater back pain reduction at 3-6 months and 2 years ( P < .05) and superior ODI improvement at 3 and 6 months ( P < .001), although long-term ODI scores were similar. Radiographically, OLIF provided greater restoration of disc height and segmental lordosis (all P < .001) and demonstrated progressive CSA increase (dynamic decompression), whereas TLIF achieved immediate, sustained CSA enlargement. Fusion rates were comparable at 1-2 years. Complication rates were low and similar (7.3% TLIF vs 5.5% OLIF), with most OLIF-specific events transient. CONCLUSION: Both OLIF and TLIF yield improvements in severe lumbar stenosis. OLIF offers perioperative advantages, earlier functional recovery, radiographic restoration, and dynamic canal remodeling, supporting its role as an equivalent alternative for lumbar spinal stenosis with some secondary advantages.

Humans

Involvement of spectrin in membrane fusion: induction of fusion in human erythrocyte ghosts by proteolytic enzymes and its inhibition by antispectrin antibody.

In contrast to intact human erythrocytes, human erythrocyte ghosts can be agglutinated but not fused by Sendai virus. Membrane fusion can, however, be induced in virus-agglutinated erythrocyte ghosts by addition of proteolytic enzymes such as trypsin, papain, or Pronase. When erythrocyte ghosts were reacted with antispectrin antiserum, the antiserum inhibited both the induction of fusion and the proteolysis of the membrane spectrin. The correlation between the membrane fusion process and the membrane cytoskeleton is discussed.

Actins

Studies of membrane fusion. III. Fusion of erythrocytes with polyethylene glycol.

Freeze-fracture electron microscopy has been used to investigate the mechanism of polyethylene glycol-induced cell fusion. Interaction of cells with the high concentrations of polyethylene glycol required for cell fusion results in cell agglutination with large planar areas of very close contact between adjacent cell membranes. An aggregation of intramembrane particles into large patches at the sites of cell-cell contact accompanies cell agglutination. Fusion occurs following the removal of most of the PEG when cells only remain in close contact at small (approximately 0.1 micrometer diameter) plaques of smooth membrane resulting in cells connected by one (or more) small cytoplasmic connexions. Expansion to form spherical fused cells occurs by a process of cell swelling.

Animals

Characterization of fusions between the lac operon and the ilv gene cluster in Escherichia coli: ilvC-lac fusions.

By means of the general procedure of Casadaban (J. Mol. Biol. 104: 541-556, 1976), the lac genes carried on a lambda-Mu-1 hybrid phage were inserted into a temperature-inducible Mu-1 prophage that had earlier been inserted into a site near the beginning of the ilvC gene of Escherichia coli strain K-12. Selection of temperature-resistant derivatives of the lysogen resulted in a fusion of the lac genes to a region of deoxyribonucleic acid that is transcribed under the control of the ilvC regulatory elements. A strain bearing the fusion was shown to be inducible for beta-galactosidase by acetohydroxybutyrate, a natural inducer of acetohydroxy acid isomeroreductase. Induction of the lysogen by mitomycin C led to the isolation of a plaque-forming lambda derivative carrying this ilvC-lac fusion.

Bacteriological Techniques

Reconstitution of cells by fusion of cell fragments. I. Myogenic expression after fusion of minicells from rat myoblasts (L6) with mouse fibroblast (A9) cytoplasm.

Rat L6 myoblasts and mouse A9 fibroblasts (HGPRT-) were enucleated by centrifugation of monolayers in the presence of cytochalasin B. Intact cells were reconstituted by Sendai virus mediated fusion of nuclei (minicells) from enucleated rat myoblasts and cytoplasms from enucleated mouse fibroblasts. Colonies arising from proliferating reconstituted cells were distinguished from intact parental cell types on the basis of nuclear and cytoplasmic markers. In five replicate experiments, approx. 70% of all colonies found after fusion were derived from reconstituted cells, 30% arose from intact rat myoblasts contaminating the minicell preparations, and two colonies were identified as hybrids between the parental cell types. Clones derived from reconstituted cells formed myotubes which produced myosin and developed the cross-striated pattern typical of skeletal muscle. The myogenic program of the rat myoblast thus can persist through the enucleation and reconstitution procedures, and is not obviously altered by a period of exposure to mouse fibroblast cytoplasm.

Animals