Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus).

Fusion of cells mediated by HVJ was inhibited completely with 5 μg/ml or more of cytochalasin D (CD). With cytochalasin, HVJ-cell interaction at 0 °C proceeded as well as without cytochalasin; HVJ was adsorbed to cell surfaces and the cells agglutinated together. Then the virus particles were enfolded with cell membranes, which resulted in the disappearance of hemadsorption activity on the cell surfaces. When the cell-virus complex was incubated at 37 °C, the early reactions proceeded as well as without cytochalasin; the hemadsorption activity reappeared on the cell surfaces, the viral envelopes fused with cell membranes at the same degree as without cytochalasin, and a stage sensitive to sodium azide appeared as in a control without cytochalasin. But cell-to-cell fusion did not occur in the presence of cytochalasin; cells were dissociated freely from the cell aggregates during incubation. This indicates that cell-to-cell fusion was inhibited but HVJ envelope to cell membrane interactions proceeded well on incubation at 37 °C. These findings suggest that viral envelope-cell membrane fusion and cell-cell fusion are separable, and participation of a cytoskeleton system including microfilaments in the cells is essential for cell-cell fusion.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus).

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Morphological changes in Ehrlich ascites tumor cells during the cell fusion reaction with HVJ (Sendai virus). III. Morphological characterization of HVJ glycoproteins integrated into the plasma membrane and their internalization by coated vesicles

On cell-cell fusion of Ehrlich ascites tumor (EAT) cells with HVJ, HVJ envelopes also fuse with the cell membrane, resulting in integration of the viral envelope glycoproteins into the fused cell membranes. Morphological characterization of the glycoproteins in the plasma membrane and the mode of their internalization were investigated in detail. In the fusion reaction, the glycoproteins were rapidly integrated into the cell membrane within 2 or 3 min on incubation at 37 °C and they remained at the fusion sites, not dispersing widely, during further incubation. Thus they were still present in clusters in the plasma membrane at the end of the fusion reaction. On culture of fused cells in culture medium, internalization of the viral glycoproteins was initiated by formation of coated vesicles and most of the integrated glycoproteins were endocytosed into the cytoplasm within 30 min. Soon after internalization, the coated vesicles fused with each other, losing their coat materials. The intact virions that remained unfused on the cell surface were also internalized, but coat materials did not appear on the inside surface of the cell membrane, unlike in the case of integrated glycoproteins.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : II. Effects of pretreatment with a small number of HVJ

Ehrlich ascites tumor cell membranes were completely modified after incubation at 37 °C for 30 min with a small dose of HVJ (about 0.7% of the maximum number of the virus particles that could be adsorbed onto the cells). After this treatment, the cells could adsorb further added HVJ onto their surfaces at 0 °C. But the cell agglutination which was induced by viral adsorption at 0 °C was very weak, and the interaction of the adsorbed virus with the lipid layer of the cell membrane at 37 °C preceding fusion or lysis of the cells was not strong. A discrepancy was observed between acquisition of the modification and liberation of sialic acid (destruction of viral receptors) by viral neuraminidase. The modification proceeded well on incubation at 37 °C but not at lower temperatures. The possibility that the modification is induced by fusion of viral envelopes with cell membranes is discussed.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : I. Interaction between cell membranes and virus in the early stage

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Characteristics of motor apparatus in testicular, epididymal and ejaculated spermatozoa. A study using demembranated sperm models

Morphological alterations of cytoplasmic organelles occur in the early stage of the cell fusion reaction induced by HVJ (Sendai virus) and these alterations are reversed in the late stage of the reaction. When the cells were treated with the virus at 37 °C for 3 min, the mitochondria became condensed, the cisternae of the endoplasmic reticula and Golgi stacks became highly distended, the inner space of the nuclear envelopes became enlarged, and the arrangement of 100 Å filaments became disordered. All these morphological changes were gradually reversed during further incubation at 37 °C and had been completely reversed by the end of the cell fusion reaction. These changes occurred at the time of interaction of the cell membrane with HVJ, and seemed to represent functional responses of the living cell to perturbation of the membrane resulting from its interaction with the virus.     

Fusion of Madin-Darby canine kidney cells by HVJ (Sendai virus): absence of direct association of virus particles with the site of membrane fusion

The process of cell fusion of Madin-Darby canine kidney (MDCK) cells by HVJ (Sendai virus) was investigated to determine whether the HVJ particles were directly associated with the site of membrane fusion. Confluent monolayer cultures of MDCK cells are sealed together by tight junctions on the apices of their lateral membranes, so added virus particles can be adsorbed only to the apical surfaces of the cells. After incubation with HVJ at 37 degrees C for 30 min, the cells still appeared mononucleate and unfused by light microscopy, but electron microscopic examination showed that fusion at the lateral membranes had occurred below the tight junctions. Furthermore, when fluorescein isothiocyanate (FITC)-labeled macromolecules, which cannot pass across the gap junctions, were injected into the cells at this stage, labeled macromolecules were found to diffuse into the adjacent cells. These findings strongly suggest that cell fusion was initiated in the lateral membrane, a region distinct from the site of adsorbed HVJ particles. Thus, the virus particles were not directly associated with the fusion site, but induced fusion of the lateral membranes indirectly.     

Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation

To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.     

Influence of temperature, cholesterol, dipalmitoyllecithin and Ca2+ on the rate of muscle cell fusion

The rate of muscle cell fusion increases between 28 °C and 40 °C by a factor of 15 to 20. The formal activation energy of the fusion process changes abruptly at about 35 °C. This change is discussed in terms of a phase transition of the membrane lipids at 35 °C. In the presence of cholesterol or dipalmitoyllecithin the fusion rate decreases markedly. Increasing the temperature reverses the effects of cholesterol and dipalmitoyllecithin. These results are discussed in terms of interactions between membrane lipids.     

Maintaining mosquito cell lines at high tempetatures: Effects on the replication of flaviviruses

Summary The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32°C to 34°C,Anopheles stephensi cell line could be maintained at 37°C. At higher temperatures initial growth rate was higher, but yield of cells after about a week of incubation was lower than at the standard temperature (28°C). Replication of several flaviviruses inAedes albopictus cell cultures adapted to 34.5°C was faster, and viral titers were higher than at 28°C.     

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

Tetracaine modifies the fragmentation mode of heated human erythrocytes and can induce heated cell fusion

It is known that human erythrocytes in saline fragment by development of an unstable surface wave on the cell rim when cells are heated through the denaturation temperature of the structural protein, spectrin. Here the influence of tetracaine on the fragmentation process has been recorded and analysed by video microscopy of cells heated in rectangular glass microcapillaries. The number of waves per cell rim decreases with increasing tetracaine concentration until, at 0.5 mM tetracaine, wave growth on the cell rim is suppressed on most cells and the cells internalize membrane at the cell dimple. The rate constant for the change in the number of waves per cell with increasing tetracaine concentration is 9.6 mM^?1 at a heating rate of 0.5 K/s. 50% of heated cells internalize membrane at 0.14 mM tetracaine. When cells are heated rapidly in suspension in test tubes the presence of tetracaine reduces the temperature for 50% haemolysis from 66°C for washed control cells to 60.5°C for cells in 2 mM tetracaine. Cells heated in microcapillaries in tetracaine concentrations of 3 mM and higher begin to swell before the spectrin denaturation temperature is reached. Cell fusion was observed at and above the spectrin denaturation temperature in cells heated in 3 and 4 mM tetracaine. It was also noted that the morphology of erythrocytes maintained in 3.6 mM tetracaine for times up to 30 min at 37°C or 20°C was strongly dependent on temperature and time.     

Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study.

Using a quantitative EM autoradiographic technique, we have visualized the membrane binding and receptor-mediated uptake of low density lipoprotein (LDL) in human fibroblasts. The initial binding was restricted to the plasma membrane (2 h of incubation at 4 °C) and approx. 62% of the grains could be localized to coated pits in the plasma membrane. When the incubations were carried out at 37 °C, ¹²⁵I radioactivity was found both on the membrane and within the cell and predominantly localized on or within lysosomes. In cells from the patient J. D., a familial hypercholesterolemic homozygote with an internalization defect, initial binding of ¹²⁵I-LDL was restricted to the plasma membrane but not preferentially localized to coated segments of the plasma membrane. After incubation for 30 min at 37 °C, the membrane bound ¹²⁵I-LDL in J. D. cells was not internalized. These data confirm results obtained with ferritin-labeled LDL and illustrate the complementary application of two different morphologic probes, each of which offers special advantages for special problems.     

Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly.

The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.     

Chemically induced fusion of fresh human erythrocytes.

The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to protein-free areas of lipids. Where these protein-free areas are in close contact fusion may occur.     

Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation

The transport kinetics of the influenza virus hemagglutinin from its site of synthesis to the apical plasma membrane of Madin-Darby canine kidney cells, a polarized epithelial cell line, were studied by a sensitive tryptic assay. Hemagglutinin acquired terminal sugars, as judged by sensitivity to endo-β-N-acetylglucosaminidase H, 10–15 min after synthesis, and first appeared on the apical domain 15 min later. None of the pulse-labeled hemagglutinin accumulated on the basolateral domain. At 20°C, terminal glycosylation continued, but no hemagglutinin was detected on the cell surface within 2 hr. If the incubation temperature was raised from 20°C to 37°C, hemagglutinin was quickly externalized, demonstrating that the inhibition at low temperature was reversible.     

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37°C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37°C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37°C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25°C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Difference in capacities for virion-to-virion fusion of young and aged HVJ (Sendai virus): A model of membrane fusion

Summary Young and aged HVJ virions differ structurally and morphologically due to changes that occur during aging in vitro or in ovo. Young virions soon after their budding off are rodshaped, rigid and relatively uniform in size, whereas virions that have aged in vitro after their formation are round, nonrigid and variable in size. These changes during aging seem to be due to the variation of M protein, a skeletal protein that is associated with both the envelope membrane proteins and nucleocapsid strands in the virions. The capacities for virion-to-virion fusion of young and aged virions were compared to clarify the relation between the membrane fusion and membrane-associating skeletal proteins. On treatment with polyethylene glycol (PEG), aged virions readily fused, forming large virion vesicles, but young virions were resistant to fusion. Further, aged virions fused even on incubation at 37°C without the fusogen. Thus the capacity for virion-to-virion fusion evidently increases during aging of virions. This result suggests that skeletal proteins associating with the biological membrane are important for preventing membrane fusion, and that virion-to-virion fusion is a good model system for use in studies on the mechanism of membrane fusion.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.