Sythesis of tropomyosin in cultures of differentiating muscle cells

The accumulation of tropomyosin in cultures of differentiating muscle cells was quantitatively measured. Tropomyosin was isolated from cultured cells during and after myoblast fusion; both alpha- and beta- subunits were present in myotube cultures. During fusion small amounts of tropomyosin were detectable, but, as fusion approached a maximum, tropomyosin accumulation began to increase. The increased synthesis of tropomyosin after the initiation of muscle cell fusion is consistent with the increased synthesis of other proteins characteristic of muscle, including myosin.     

Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".     

重组蛋白转导域-神经肽Y融合蛋白对体外培养大鼠脂肪细胞的影响

目的探讨重组蛋白转导域-神经肽Y融合蛋白对体外培养大鼠脂肪细胞的影响。方法采用剪切消化法分离大鼠前脂肪细胞,培养液中添加重组PTD-NPY融合蛋白,检测前脂肪细胞和成熟脂肪细胞的形态学变化、细胞中甘油三酯含量和甘油磷酸脱氢酶(GPDH)活性。结果重组PTD-NPY融合蛋白处理大鼠前脂肪细胞72 h,细胞体积增大,细胞数量增加,细胞甘油三酯含量和GPDH活性升高;重组PTD-NPY融合蛋白处理成熟脂肪细胞48h后,细胞体积明显增大,细胞内脂肪滴数量增加并融合成较大的脂滴,甘油三酯含量和GPDH活性均显著升高。结论重组PTD-NPY融合蛋白明显促进前脂肪细胞的分化,促进脂肪细胞中甘油三酯的合成与沉积。为重组PTD-NPY融合蛋白在动物生产及人类疾病治疗中的实际应用奠定理论基础。     

Effects of divalent cations,temperature, osmotic pressure gradient,and vesicle curvature on phosphatidylserine vesicle fusion

Summary Fusion of phosphatidylserine vesicles induced by divalent cations, temperature and osmotic pressure gradients across the membrane was studied with respect to variations in vesicle size. Vesicle fusion was followed by two different methods: 1) the Tb/DPA fusion assay, whereby the fluorescent intensity upon mixing of the internal aqueous contents of fused lipid vesicles was monitored, and 2) measurement of the changes in turbidity of the vesicle suspension due to vesicle fusion. It was found that the threshold concentration of divalent cations necessary to induce vesicle fusion depended on the size of vesicles; as the diameter of the vesicle increased, the threshold value increased and the extent of fusion became less. For the osmotic pressure-induced vesicle fusion, the larger the diameter of vesicles, the smaller was the osmotic pressure gradient required to induce membrane fusion. Divalent cations, temperature increase and vesicle membrane expansion by osmotic pressure gradient all resulted in increase in surface energy (tension) of the membrane. The degree of membrane fusion correlated with the corresponding surface energy changes of vesicle membranes due to the above fusion-inducing agents. The increase in surface energy of 9.5 dyn/cm from the reference state corresponded to the threshold point of phosphatidylserine membrane fusion. An attempt was made to explain the factors influencing fusion phenomena on the basis of a single unifying theory.     

Inhibition of Akt activity and calcium channel function coordinately drive cell-cell fusion in the BeWO choriocarcinoma placental cell line

To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and concentration dependent manner consistent with the increased secretion of beta human chorionic gonadotrophin (β-HCG) and appearance of multi-nucleated cells. Analyses of the fusion process demonstrated that in addition to increased cAMP levels, simultaneous reduction of intracellular calcium and inhibition of Type 1 phosphatidylinositol 3 kinase (PI3K)/Akt signaling also resulted in cell fusion. Although individual blockade of calcium channel function or PI3K/Akt signaling was without effect, the combination with forskolin resulted in a potentiation of cell fusion. These data demonstrate syncytialization is a complex process that depends upon the regulation of distinct signaling inputs that function in concert with each other.     

In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion

The role of cell volume in regulating cell fusion was investigated by in vitro fertilization to elucidate mechanisms involved in double fertilization. The results revealed that in our model gamete fusion was more efficient than the fusion of somatic cells under the same conditions. The fusion of selected mesophyll protoplasts of different sizes and their fusion with chloroplasts demonstrated that cell volume ratio played a role in this process: as the ratio increased, fusion was more efficient and faster. When one of the cells was as small as a sperm, the formation of a round fusion product was faster. This might explain why gamete fusion was highly efficient in all in vitro germ cell fusion systems. This finding may also explain why sperm evolved as small cells. The results reported here will be useful for interpreting and evaluating data of in vitro fertilization experiments and for distinguishing gamete-specific characters. Received: 12 September 2000 / Revision accepted: 6 December 2000     

A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli

The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.     

Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion.

Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.     

Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.     

Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum-starved skin cells were electrofused to enucleated oocytes, activated 1-5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1-2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6-56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus-like structure (PN), whereas, 64.5-78.3% of embryos activated 1-2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3-21.7%) compared to those of others (0-8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos.     

Free fatty acid enhancement of cation-induced fusion of liposomes: synergism with synexin and other promoters of vesicle aggregation

The effect of free fatty acids on the cation-induced fusion of large unilamellar vesicles (liposomes) was investigated by using fluorescent assays which monitor the mixing of aqueous contents of liposomes. Overall fusion was modeled as a two-step process involving aggregation of vesicles followed by actual fusion. Different experimental conditions were used which favored either aggregation or fusion as the rate-limiting step in the overall process. When phosphatidylserine liposomes were induced to fuse by 4 mM Ca2+ plus 5 mM Mg2+, preincubation with arachidonic acid showed a dramatically increased overall rate of fusion compared to the same liposomes not treated with fatty acid. When fusion was induced by 3 mM Ca2+, arachidonic acid had little effect. These results were interpreted in terms of the action of arachidonic acid only at the fusion step per se and not the aggregation step. Therefore, the enhancement of the overall fusion rate would be observed solely under conditions where the actual fusion of liposomes was rate limiting (Ca/Mg) rather than the aggregation of liposomes (Ca alone). When other liposome systems were tested, the effect of arachidonic acid was observed only under fusion rate-limiting conditions. Arachidonic acid was found to act synergistically with promoters of liposomal aggregation, such as Mg2+, spermine, and synexin, to enhance the overall rate of liposome fusion, as would be expected from action at separate kinetic steps. The dependence of the fusion rates on arachidonic acid concentration demonstrated an apparently cooperative effect. The structure of the fatty acid is of critical importance in determining its effects, as shown by the fact that 16-doxylstearic acid always increased the rate of fusion while 5-doxylstearic acid always decreased the rate of fusion under all conditions tested. A number of different fatty acids, including oleic acid, elaidic acid, 16-doxylstearic acid, myristic acid, and stearic acid, were effective at increasing the fusion rate to varying extents. In general, unsaturated fatty acids were more effective than saturated ones, either due to partitioning into the membrane or because of structural requirements for promotion of fusion.     

Control of membrane fusion mechanism by lipid composition: predictions from ensemble molecular dynamics

Membrane fusion is critical to biological processes such as viral infection, endocrine hormone secretion, and neurotransmission, yet the precise mechanistic details of the fusion process remain unknown. Current experimental and computational model systems approximate the complex physiological membrane environment for fusion using one or a few protein and lipid species. Here, we report results of a computational model system for fusion in which the ratio of lipid components was systematically varied, using thousands of simulations of up to a microsecond in length to predict the effects of lipid composition on both fusion kinetics and mechanism. In our simulations, increased phosphatidylcholine content in vesicles causes increased activation energies for formation of the initial stalk-like intermediate for fusion and of hemifusion intermediates, in accordance with previous continuum-mechanics theoretical treatments. We also use our large simulation dataset to quantitatively compare the mechanism by which vesicles fuse at different lipid compositions, showing a significant difference in fusion kinetics and mechanism at different compositions simulated. As physiological membranes have different compositions in the inner and outer leaflets, we examine the effect of such asymmetry, as well as the effect of membrane curvature on fusion. These predicted effects of lipid composition on fusion mechanism both underscore the way in which experimental model system construction may affect the observed mechanism of fusion and illustrate a potential mechanism for cellular regulation of the fusion process by altering membrane composition.     

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.     

Membrane fusion correlates with surface charge in exocytic vesicles

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.     

Directed evolution of a novel N-carbamylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability

Bifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial fusion enzymes remains to be solved. Based on structural traits of microbial D-hydantoinase, we attempted to construct a bifunctional N-carbamylase/D-hydantoinase fusion enzyme that would be useful for the synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain polypeptide in E. coli. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11, was selected after repeated rounds, and this enzyme was found to show sixfold increased performance in the production of D-amino acid compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential.     

Superior protective and therapeutic effects of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma

Fusion vaccine of dendritic cells (DCs) and tumor cells has the advantage of inducing an immune response against multiple tumor Ags, including unknown tumor Ags. Using the liver metastasis model of C1300 neuroblastoma cells, we assessed the protective and therapeutic effects of fusion cells transduced with the IL-12 gene and/or the IL-18 gene. Improving the fusion method by combining polyethylene glycol and electroporation increased loading efficiency. In the A/J mice vaccinated with fusion cells modified with the LacZ gene (fusion/LacZ), IFN-gamma production and CTL activity increased significantly compared with that of DCs/LacZ, C1300/LacZ, or a mixture of the two (mixture/LacZ). With the transduction of IL-12 and IL-18 genes into the fusion cells (fusion/IL-12/IL-18), the level of IFN-gamma increased more than five times that of other fusion groups. In addition, NK cell activity and CTL activity increased significantly compared with that of mixture/LacZ, fusion/LacZ, DC/LacZ, or C1300/LacZ. In the protective and therapeutic studies of fusion cell vaccine, mice vaccinated with fusion/LacZ, fusion/IL-12, fusion/IL-18, or fusion/IL-12/IL-18 showed a significant decrease in liver metastasis and a significant increase in survival compared with mice given a mixture/LacZ, DCs/LacZ, or C1300/LacZ. In particular, the mice receiving fusion/IL-12/IL-18 vaccine showed a complete protective effect and the highest therapeutic effects. The present study investigates the improved loading efficiency of fusion cells and suggests that the introduction of IL-12 and IL-18 genes can induce extremely strong protective and therapeutic effects on liver metastasis of neuroblastoma.     

Membrane cholesterol and cell fusion of hen and guinea-pig erythrocytes.

1. The cholesterol content of hen erythrocytes was modified by treating the cells with phospholipid liposomes. 2. Depletion of cellular cholesterol, by using liposomes of dipalmitoylglycerophosphocholine or phosphatidylcholine from hen erythrocytes, had no effect on the susceptibility of the cells to fusion induced by oleoylglycerol, but markedly decreased fusion induced by Sendai virus. 3. By contrast, enrichment of cellular cholesterol by using liposomes of dipalmitoylglycerophosphocholine and cholesterol increased cell fusion induced by oleoylglycerol, poly(ethylene glycol) and Sendai virus. 4. Virus-induced cell fusion of guinea-pig erythrocytes, which were enriched in cholesterol by feeding a cholesterol-rich diet to the animals, was also enhanced. 5. Hen erythrocytes that were treated with liposomes prepared from egg phosphatidylcholine contained increased quantities of phospholipid phosphorus and fused readily on incubation with retinol, independently of their cholesterol content. 6. It is suggested that cholesterol may enhance cell fusion by acting to facilitate a phase separation of protein-free areas of lipid bilayer, which subsequently provide the sites for cell fusion.     

Apport de la fusion d’images tomoscintigraphiques avec la tomodensitométrie X ou l’imagerie par résonance magnétique en pathologie osseuse : expérience du service de médecine nucléaire de Sfax

The aim of our study was to assess the contribution of image fusion of SPECT combined with computed tomography (SPECT-CT) or magnetic resonance imaging (SPECT-MRI) in our daily practice of bone scintigraphy. Seventeen patients underwent a SPECT image fusion, SPECT-CT or MRI in addition to conventional bone scintigraphy. These acquisitions are made separately with images stored in DICOM format. The fusion was operated after a manual coregistration of the images. Results show that in 35.29% of cases, the image fusion allowed to pinpoint the exact location of increased uptake seen on the whole body scan and/or on static images. In 35.29% of cases, the fusion has confirmed a diagnosis doubtful in planar imaging. In five patients, representing 29.41% of cases, the fusion has corrected a diagnosis. In one patient (5.88% of cases), the fusion has eliminated a false positive related to increased uptake visualized on the whole body scan. In two patients (11.11% of cases), the fusion has eliminated false negative of bone scan in one case and of MRI in the other case. Finally, in nine patients (50% of cases), the fusion has influenced the therapeutic management.     

Effects of fusion temperature on the lateral mobility of Sendai virus glycoproteins in erythrocyte membranes and on cell fusion indicate that glycoprotein mobilization is required for cell fusion

In order to investigate the requirement for lateral mobilization of viral envelope glycoproteins on the cell surface in the induction of cell-cell fusion, we employed fluorescence photobleaching recovery to study the effect of the fusion temperature on the lateral mobilization of Sendai virus glycoproteins in the human erythrocyte membrane. As the fusion temperature was reduced below 37 degrees C (to 31 or 25 degrees C), the rates of virus-cell fusion, the accompanying hemolysis, and cell-cell fusion were all slowed down. However, the plateau (final level) after the completion of fusion was significantly reduced at lower fusion temperatures only in the case of cell-cell fusion, despite the rather similar final levels of virus-cell fusion. A concomitant decrease as a function of the fusion temperature was observed in the fraction of cell-associated viral glycoproteins that became laterally mobile in the erythrocyte membrane during fusion, and a strict correlation was found between the level of laterally mobile viral glycoproteins in the cell membrane and the final extent of cell-cell fusion. The accompanying reduction in the lateral diffusion coefficients (D) of the viral glycoproteins (1.4-fold at 31 degrees C and 1.9-fold at 25 degrees C, as compared to 37 degrees C) does not appear to determine the final level of cell-cell fusion, since fusing the cells with a higher amount of virions at 25 degrees C increased the final level of cell-cell fusion while D remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of cyclic GMP in the fusion of chick embryonic myoblasts in culture.

We found that a transient rise in cGMP levels, which was closely associated with the Ca2+ influx, occurred concomitant with the onset of myoblast fusion. The Ca2+ channel blocker D600 decreased both the cell fusion and the normal rise in cGMP levels. In contrast, the Ca2+ ionophore A23187 transiently increased cGMP levels and induced precocious fusion. In addition, the cGMP analog 8-Br-cGMP induced precocious fusion as A23187 did. The guanylate cyclase inhibitor, methylene blue delayed the fusion in a dose-dependent manner without significantly affecting cell alignment, proliferation, or muscle-specific protein expression. Furthermore, methylene blue delayed the normal rise in cGMP levels, and the fusion block imposed by methylene blue was significantly recovered by 8-Br-cGMP. On the basis of our present findings, we suggest that a Ca2+ influx-dependent rise in cGMP levels is an important step in myoblast fusion.