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.

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.

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 Insertional Mutations in Herpes Simplex Virus Type 1 gL Identify Functional Domains for Association with gH and for Membrane Fusion

2009 ◽  
Vol 83 (22) ◽  
pp. 11607-11615 ◽  
Author(s):  
Qing Fan ◽  
Erick Lin ◽  
Patricia G. Spear
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Viral Entry ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Conserved Domain ◽  
Simplex Virus

ABSTRACT Glycoprotein L (gL) is one of four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion. This glycoprotein oligomerizes with gH to form a membrane-bound heterodimer but can be secreted when expressed without gH. Twelve unique gL linker-insertion mutants were generated to identify regions critical for gH binding and gH/gL processing and regions essential for cell fusion and viral entry. All gL mutants were detected on the cell surface in the absence of gH, suggesting incomplete cleavage of the signal peptide or the presence of a cell surface receptor for secreted gL. Coexpression with gH enhanced the levels of cell surface gL detected by antibodies for all gL mutants except those that were defective in their interactions with gH. Two insertions into a conserved region of gL abrogated the binding of gL to gH and prevented gH expression on the cell surface. Three other insertions reduced the cell surface expression of gH and/or altered the properties of gH/gL heterodimers. Altered or absent interaction of gL with gH was correlated with reduced or absent cell fusion activity and impaired complementation of virion infectivity. These results identify a conserved domain of gL that is critical for its binding to gH and two noncontiguous regions of gL, one of which contains the conserved domain, that are critical for the gH/gL complex to perform its role in membrane fusion.

scholarly journals Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion

2003 ◽  
Vol 77 (1) ◽  
pp. 499-510 ◽  
Author(s):  
Timothy P. Foster ◽  
Xavier Alvarez ◽  
Konstantin G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Membrane Topology ◽  
Cell Surfaces ◽  
Glycoprotein K ◽  
Simplex Virus

ABSTRACT Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gK-transfected cells with the gK-null virus ΔgK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.

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.

Effect of Solution Osmotic Pressure on Cell Fusion by Poly(Ethylene Glycol)

1995 ◽  
Vol 10 (1) ◽  
pp. 14-27 ◽  
Author(s):  
Naoki Nakajima ◽  
Yoshito Ikada
Keyword(s):  
Membrane Fusion ◽  
Osmotic Pressure ◽  
Cell Fusion ◽  
Culture Medium ◽  
Morphological Changes ◽  
Cell Swelling ◽  
Cell Incubation ◽  
Peg Treatment ◽  
Poly Ethylene ◽  
Osmotic Pressure Difference

Effects of the osmotic pressure of culture medium on the membrane fusion of L929 cells in the monolayer state were investigated using polyethylene glycol) (PEG) with the molecular weight of 3,000 at various concentrations at phosphate buffer saline (PBS). Cell incubation for fusion was performed via three stages; (1) incubation before PEG treatment (preincubation), (2) incubation in the presence of PEG (PEG incubation), and (3) incubation after PEG treatment (postincubation). The PBS concentrations half that of a isotonic solution in the pre- and postincubation stages significantly accelerated the membrane fusion, whereas cell treatment at more hypotonic or hypertonic concentrations of PBS suppressed cell fusion. This result was explained in terms of cell swelling and shrinking induced by the osmotic pressure difference, because such cell morphological changes actually occurred when the PBS concentration was varied from the isotonicity. In contrast, almost no effect of osmotic pressure on cell fusion was observed if PEG was present in the culture medium at 40 w/w% concentration, regardless of the PBS concentration.

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.

scholarly journals Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells.

1989 ◽  
Vol 37 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Z Z Zou ◽  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Tight Junctions ◽  
Milk Fat ◽  
Freeze Fracture ◽  
Ruthenium Red ◽  
Single Cell Suspension ◽  
Monolayer Cultures ◽  
Milk Fat Globule ◽  
Entire Cell ◽  
Fat Globule ◽  
Mcf 7

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).

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

1979 ◽  
Vol 36 (1) ◽  
pp. 73-84
Author(s):  
S. Knutton
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Hela Cells ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Plasma Membrane ◽  
Virus Particles ◽  
Cell Plasma

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.

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.

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