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

1979 ◽  
Vol 36 (1) ◽  
pp. 61-72
Author(s):  
S. Knutton
Keyword(s):  
Polyethylene Glycol ◽  
Cell Fusion ◽  
Close Contact ◽  
Freeze Fracture ◽  
Cell Swelling ◽  
Adjacent Cell ◽  
Cell Contact ◽  
Cell Agglutination ◽  
High Concentrations ◽  
Freeze Fracture Electron Microscopy

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.

scholarly journals Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes.

1983 ◽  
Vol 96 (1) ◽  
pp. 151-159 ◽  
Author(s):  
J W Wojcieszyn ◽  
R A Schlegel ◽  
K Lumley-Sapanski ◽  
K A Jacobson
Keyword(s):  
Polyethylene Glycol ◽  
Cell Fusion ◽  
Cultured Cells ◽  
Membrane Lipids ◽  
Freeze Fracture ◽  
Water Soluble ◽  
Erythrocyte Membranes ◽  
Adjacent Cell ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.

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

1977 ◽  
Vol 28 (1) ◽  
pp. 179-188
Author(s):  
S. Knutton ◽  
D. Jackson ◽  
M. Ford
Keyword(s):  
Cell Surface ◽  
Cell Fusion ◽  
Hela Cells ◽  
Cell Swelling ◽  
Cell Contact ◽  
Cell Agglutination ◽  
Scanning Electron Microscope Study ◽  
Virus Particles ◽  
Scanning Electron ◽  
Cell Cell

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.

Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion

1980 ◽  
Vol 43 (1) ◽  
pp. 103-118
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Fusion Event ◽  
Virus Envelope ◽  
Membrane Rupture ◽  
Membrane Tubules

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

Cell hybridization and cell agglutination. I. Enhancement of cell hybridization by lectins

1985 ◽  
Vol 78 (1) ◽  
pp. 263-271
Author(s):  
Y. Matsuya ◽  
I. Yamane
Keyword(s):  
Polyethylene Glycol ◽  
Cell Fusion ◽  
Wheat Germ ◽  
Great Increase ◽  
Cell Hybridization ◽  
Cell Agglutination ◽  
Plant Lectins ◽  
Peg Treatment ◽  
A Cell ◽  
Conventional Cell

A great increase in hybridization frequency of cultured rodent cells was obtained when conventional cell fusion using 50% polyethylene glycol (PEG) was combined with a cell agglutination produced by plant lectins. The rate of appearance of hybrid colonies was found to be correlated with the extent of cell agglutination by lectin, as well as with cell fusion induced by subsequent PEG treatment. Phytohemagglutinin (PHA), wheat germ agglutinin, Wistaria floribunda agglutinin and concanavalin A were all active; the most effective was PHA. When parental cells in a monolayer were treated with PHA followed by PEG, the resulting hybridization frequency was very low because of markedly decreased viability, whereas the same cells in suspension yielded hybrid colonies at a higher rate. These results suggest that the enhancement of hybridization by PHA/PEG treatment was brought about by the ability of lectin to agglutinate cells.

scholarly journals Membrane differentiations at sites specialized for cell fusion.

1977 ◽  
Vol 72 (1) ◽  
pp. 144-160 ◽  
Author(s):  
R L Weiss ◽  
D A Goodenough ◽  
U W Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Active Role ◽  
Central Dome ◽  
Thin Sections ◽  
Mating Structure ◽  
Putative Site ◽  
Freeze Fracture Electron Microscopy

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

Discrimination of Two Fusogenic Properties of Aqueous Polyethylene Glycol Solutions

1981 ◽  
Vol 36 (7-8) ◽  
pp. 593-596 ◽  
Author(s):  
Hermann Krähling
Keyword(s):  
Polyethylene Glycol ◽  
Cell Line ◽  
Cell Fusion ◽  
Mammalian Cell ◽  
Dose Response ◽  
Cell Contact ◽  
Cell Shrinkage ◽  
Mammalian Cell Lines ◽  
A Cell ◽  
Free Membrane

Abstract Investigations on the dose response of cell fusion, induced by ionfree aqueous polyethylene glycol (PEG) solutions, reveal distinct lowest fusogenic PEG concentrations for different permanently growing mammalian cell lines. Part of the requisite PEG can be replaced by carbo­ hydrates, preserving the fusogenity of the solutions. This discriminates two effects of PEG solutions causing cell fusion: a) cell shrinkage, the required hyperosmolality of the solutions may be provided by PEG or by carbohydrates, is supposed to cause intracellular processes necessary for consolidating polycaryons; b) membrane alterations, which can not be induced by carbo­ hydrates, enable intimate cell-cell contact via particle-free membrane areas. Depending on cell line salts can not only raise the osmolality of PEG solutions but are able to co-operate with PEG in generating membrane alterations.

Membrane alterations and other morphological features associated with polyethylene glycol-induced cell fusion

1979 ◽  
Vol 40 (1) ◽  
pp. 63-75
Author(s):  
J.M. Robinson ◽  
D.S. Roos ◽  
R.L. Davidson ◽  
M.J. Karnovsky
Keyword(s):  
Polyethylene Glycol ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Deleterious Effect ◽  
Freeze Fracture ◽  
Neighbouring Cell ◽  
Monolayer Cultures ◽  
Peg Treatment ◽  
Entire Cell

Polyethylene glycol (PEG) induces rapid fusion of LM cells. Membrane fusion, as detected by formation of pentalaminar membrane arrays, occurs as early as 1 min after PEG treatment. The entire cell surface arrears to be capable of fusion since fusion occurs in regions where pseudopodia make contact with each other or with a neighbouring cell body and also in areas where cells are in contact along their entire periphery. Cytoskeletal components showed no apparent deleterious effect from PEG treatment or subsequent cell fusion as determined by thin-section EM. Freeze-fracture of monolayer cultures reveals a thermotropic rearrangement of intramembranous particles following PEG treatment.

scholarly journals Cell hybridization and cell agglutination. II. Enhancement of cell hybridization by polycations

1985 ◽  
Vol 78 (1) ◽  
pp. 273-282
Author(s):  
Y. Matsuya ◽  
I. Yamane
Keyword(s):  
Polyethylene Glycol ◽  
Cell Fusion ◽  
Mammalian Cells ◽  
Polyvinyl Pyrrolidone ◽  
Efficient Technique ◽  
Cell Hybridization ◽  
Cell Agglutination ◽  
Peg Treatment

An efficient technique for hybridization of mammalian cells was developed by combining agglutination by pretreatment with polycations, such as polyarginine, and conventional polyethylene glycol(PEG)-mediated cell fusion. Polyarginine and subsequent PEG treatment resulted in markedly decreased viability in the treated cells, but addition of polyvinyl pyrrolidone or glycerol to the polyarginine prevented this cytotoxicity. Polyarginine was much more effective than polylysine or polyornithine in inducing hybridization. Other polycations, including polybrene and protamine but not DEAE-dextran, were also active in inducing hybridization. The condition of the cells at the time of polycation treatment was an important factor in the enhancement of hybridization. The condition of the cells at the time of polycation treatment was an important factor in the enhancement of hybridization. The enhancement of hybridization of cells in monolayer incubated for 2 h was much higher than that of cells incubated for 24 h. These findings suggest that polycations do not necessarily operate by agglutinating cells. The mechanism of polycation-enhanced cell hybridization is discussed.

scholarly journals Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells.

1983 ◽  
Vol 97 (3) ◽  
pp. 909-917 ◽  
Author(s):  
D S Roos ◽  
J M Robinson ◽  
R L Davidson
Keyword(s):  
Polyethylene Glycol ◽  
Particle Distribution ◽  
Freeze Fracture ◽  
Visual Observation ◽  
Accurate Analysis ◽  
The Mean ◽  
Low Levels ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Resistant Cells

The distribution of intramembrane particles (IMP) as revealed by freeze-fracture electron microscopy has been analyzed following treatment of mouse L cells and fusion-deficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, greater than 90% of the cells fuse, and cold-induced IMP aggregation is extensive. In contrast, identical treatment of fusion-deficient cell lines shows neither extensive fusion nor IMP redistribution. At higher concentrations of PEG, however, the PEG-resistant cells fuse extensively and IMP aggregation is evident. Thus the decreased ability of the fusion-deficient cells to fuse after treatment with PEG is correlated with the failure of IMP aggregation to occur. A technique for quantifying particle distribution was developed that is practical for the accurate analysis of a large number of micrographs. The variance from the mean number of particles in randomly chosen areas of fixed size was calculated for each cell line at each concentration of PEG. Statistical analysis confirms visual observation of highly aggregated IMP, and allows detection of low levels of aggregation in parental cells that were less extensively fused by exposure to lower concentrations of PEG. When low levels of fusion were induced in fusion-deficient cells, however, no IMP aggregation could be detected.

Freeze-fracture, freeze-etch complementary pairs of virus-infected cells reveal value of etching even when relatively high concentrations of cryoprotectants are used

1978 ◽  
Vol 36 (2) ◽  
pp. 136-137
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Plant Tissue ◽  
Phosphate Buffer ◽  
Infected Plant ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Virus Infected Plant ◽  
Infected Cells ◽  
High Concentrations

It has been assumed by many involved in freeze-etch or freeze-fracture studies that it would be useless to etch specimens which were cryoprotected by more than 15% glycerol. We presumed that the amount of cryoprotective material exposed at the surface would serve as a contaminating layer and prevent the visualization of fine details. Recent unexpected freeze-etch results indicated that it would be useful to compare complementary replicas in which one-half of the frozen-fractured specimen would be shadowed and replicated immediately after fracturing whereas the complement would be etched at -98°C for 1 to 10 minutes before being shadowed and replicated.Standard complementary replica holders (Steere, 1973) with hinges removed were used for this study. Specimens consisting of unfixed virus-infected plant tissue infiltrated with 0.05 M phosphate buffer or distilled water were used without cryoprotectant. Some were permitted to settle through gradients to the desired concentrations of different cryoprotectants.

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