Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Surface characterization of poly(ethylene glycol) coated human red blood cells by particle electrophoresis

Recent studies have shown that the covalent attachment of poly(ethylene glycol), abbreviated as PEG, to the surface of human red blood cells (RBC) leads to masking of membrane antigenic sites and inhibition of RBC aggregation. The effects of PEG coating on the regions near the RBC glycocalyx were thus explored using cell micro-electrophoresis. Both linear (3.35, 18.5, 35.0) and an 8-arm 35.9 kDa reactive PEG were used; in one series, thick cross-linked coats were obtained using a branched PEG amine as a cross-linker. The results indicate marked decreases of RBC mobility (up to 90%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment is predominantly to primary amine groups, such decreases of mobility most likely reflect structural changes near and within the RBC glycocalyx rather than decreased surface charge density. Experimental data were analyzed using a theoretical approach which allows calculation of the thickness and friction of the polymer layers: (1) for linear PEGs, thickness increased and friction decreased with polymer mass; (2) compared to linear PEGs of similar molecular mass, thickness was less and friction was greater for the branched PEG; (3) cross-linked PEG coatings were more than 50 nm thick and were insensitive to changes of ionic strength. These observations are consistent with the aggregation behavior of PEG-coated RBC and indicate the usefulness of micro-electrophoresis methods for studies of covalently-attached polymers: the resulting calculated thickness and friction factors should be of value in achieving desired cellular surface characteristics or levels of cell-cell interaction.     

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Fluorescence microscopic study of cells and polykaryons in the early periods following polyethylene glycol treatment

A fluorescence-microscopical study is made of cultured murine fibroblasts (L-cells) in early periods after the treatment with polyethylene glycol (PEG). Optimal conditions of fusion procedure were found under which the effectiveness of fusion was the highest and the toxical effect of PEG the lowest. The number of dead cells after the treatment with PEG did not exceed 10%. No significant changes in chromatin cytochemical properties (Acridine Orange and Olivomycin binding) were observed in the early periods of PEG treatment, that allows to use PEG for studying chromatin properties in hybrid cells obtained by PEG fusion. By means of PEG fusion, the hybrid cells with prematurely condensed chromosomes and also hybrids between animal and yeast cells have been obtained.     

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol)

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.     

Chemical co-treatments and intramembrane particle patching in the poly(ethylene glycol)-induced fusion of turkey and human erythrocytes

Several chemical co-treatments were used to lower the threshold concentrations of poly(ethylene glycol) (PEG) required to induce fusion between turkey erythrocytes and between human erythrocytes. Concanavalin A was used in conjunction with 25% (w/w) PEG to induce turkey erythrocyte fusion. The fusion percentage increased with increasing concentrations of concanavalin A and the duration of concanavalin A treatment. In samples with high percentages of fusion, numerous hemispherical intramembrane particle-free zones (bubbles) in the plasma membrane were revealed by freeze-fracture electron microscopy. However, concanavalin A treatment did not facilitate fusion between human erythrocytes even at 35% PEG, although slight intramembrane particle patching was observed under this condition. Spermidine (0.05% w/v), trichloroacetic acid (100 mM) and ethanol (4% v/v) were found to promote fusion of human erythrocytes in 25% PEG. In all of these cases, intramembrane particle patching was observed by freeze-fracture electron microscopy in the presence of PEG. When applied alone, only ethanol caused a slight intramembrane particle patching. Neither dimethylsulfoxide (2% v/v), lysophosphatidylcholine (lysoPC, 0.15 mM), nor polylysine (mol. wt. 1000-4000, 0.05% w/v) promoted fusion of human erythrocyte in 25% PEG. None of these chemical treatments, alone, or in combination with PEG, caused intramembrane particle patching. We conclude that the positive effect of chemical treatments on PEG-induced cell fusion is closely related to the formation of intramembrane particle-free zones on the plasma membrane.     

Characterization of PEG-mediated electrofusion of human erythrocytes.

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Time sequence and morphological evaluations of cells fused by polyethylene glycol 6000(1).

Mouse L cells (clone 1D) were fused with polyethylene glycol (PEG). The fusion sequence was determined by using sequential light microscopy of the same group of cells, scanning electron microscopy (SEM),transmission electron microscopy, and freeze-etching. The cells were found to fuse only 1 min after PEG had been washed off at small localized areas. Larger fusion images were found after 3 min. Intramembrane particles were observed to have a tendency to aggregate after PEG treatment, but a direct correlation of this activity with the fusion process could not be made. No pathological changes were noted at longer times after PEG removal, except for the extensive widening of the rough-surfaced endoplasmic reticulum (RER) in some cells. It is proposed that fusion does not occur if apposing cells have many microvilli at the area of apparent contact.     

Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (< 4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.     

The fusion of erythrocytes by treatment with proteolytic enzymes and polyethylene glycol.

Polyethylene glycol (PEG) has been utilized to induce homokaryocyte formation in avian and mammalian erythrocytes previously treated with proteolytic enzymes. PEG of molecular weight 6,000-7,5000 was found superior to 1,500 and 20,000 MW PEG. Cells exposed to protease alone, prior to PEG treatment, fused to a high degree (60-95% multinucleated cells), whereas trypsin or pepsin treatment alone allowed very little fusion (2.5%). Trypsin lowered the effectiveness of protease when used in combination. Cells which were not treated with proteolytic enzymes agglutinated in the presence of PEG but did not fuse to a significant extent (0.01%). Fusion was also markedly dependent upon the rate at which PEG was eluted during the fusion process. Electron microscopy indicated that fusion began during the elution of PEG from the agglutinated cells.     

A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.

The yield of hybrid colonies after fusion of mammalian cells with polyethylene glycol (PEG) is increased if the cells are fused in Ca2+-free medium, and kept in Ca2+-free medium for at least 15 min after fusion. The protective effect of Ca2+-free medium is much more obvious when Baker PEG is used than when fusion is carried out with Koch-Light PEG. The increased yield of hybrid colonies is shown to be due to a reduced toxicity rather than to an increased efficiency of cell fusion. These improvements have been found to apply to a variety of cell lines, and also when cell fusion is carried out in suspension. This technique should be particularly useful in studies on mammalian cell hybridization using cell lines that are particularly sensitive to the toxic effect of PEG.     

A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.     

Kinetics of influenza hemagglutinin-mediated membrane fusion as a function of technique

Reliable techniques are required to evaluate the plausibility of proposed membrane fusion mechanisms. Here we have studied the kinetics of establishing the lipidic connection between hemagglutinin-expressing cells (HA-cells) and red blood cells (RBC) labeled with octadecylrhodamine, R18, using three different experimental approaches: (1) the most common approach of monitoring the rate of the R18 dequenching in a cuvette with a suspension of RBC/HA-cell complexes; (2) video fluorescence microscopy (VFM) to detect the waiting times before the onset of R18 redistribution, not dequenching, for each RBC attached to an adherent HA-cell; and (3) a new approach based on blockage of RBC fusion to an adherent HA-cell at different time points by lysophosphatidylcholine (LPC), so that only the cell pairs which, at the time of LPC application, had fused or were irreversibly committed to fusion contributed to the final extent of lipid mixing. The LPC blockage and VFM gave very similar estimates for the fusion kinetics, with LPC monitoring also those sites committed to the lipid mixing process. In contrast, R18 dequenching in the cuvette was much slower, i.e., it monitors a much later stage of dye redistribution.     

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.     

Cell fusion and intramembrane particle distribution in polyethylene glycol-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.     

Electron microscopic study of plant-animal cell fusion

Summary Avian erythrocytes and protoplasts isolated from mesophyll cells of tobacco plants were suspended in 1% protease, agglutinated with polyethylene glycol (PEG) and subsequently fused upon elution of the PEG. The fusion reaction was monitored by scanning (SEM) and transmission (TEM) electron microscopy. SEM studies showed a marked difference in the topography of agglutinated cells. During, and subsequent to fusion, the markedly different surfaces of the two cell types became homogeneous and lines of demarcation between the cells were no longer visible. TEM revealed that adhesion occurred over the entire membrane area between agglutinated cells. Incipient fusion was evidenced by the appearance of vacuoles at the intermembrane surfaces. During initial elution of the PEG, cytoplasmic channels between erythrocytes and protoplasts were evident. With continued elution of the PEG, starch-containing plant chloroplasts and starch grains were seen within erythrocytes and homogenous erythrocyte cytoplasm was present inside plant protoplasts. Cytoplasmic mixing between the two cell types occurred within 3 hours of elution. The frequency of interkingdom fusion was estimated to be 0.5–1%.     

Time sequence and morphological evaluations of cells fused by polyethylene glycol 6000

Summary Mouse L cells (clone 1D) were fused with polyethylene glycol (PEG). The fusion sequence was determined by using sequential light microscopy of the same group of cells, scanning electron microscopy (SEM), transmission electron microscopy, and freeze-etching. The cells were found to fuse only 1 min after PEG had been washed off at small localized areas. Larger fusion images were found after 3 min. Intramembrane particles were observed to have a tendency to aggregate after PEG treatment, but a direct correlation of this activity with the fusion process could not be made. No pathological changes were noted at longer times after PEG removal, except for the extensive widening of the rough-surface endoplasmic reticulum (RER) in some cells. It is proposed that fusion does not occur if apposing cells have many microvilli at the area of apparent contact. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This work was supported by U.S. Public Health Service research grants CA 10815 from the National Cancer Institute and GM 21615 from the Institute of General Medical Sciences.     

F-actin is involved in the polyethylene glycol-induced fusion of chick embryo fibroblasts

Cytochalasin B inhibits the polyethylene glycol (PEG)-induced fusion of chick embryo fibroblasts. Induction of fusion of these cells by PEG is associated with transient changes in the pattern of F-actin organization within the cell. The changes include the disappearance of stress fibres and accumulation of F-actin under the plasma membrane. These results suggest an involvement of F-actin in PEG-induced cell fusion.