A Quantitative Fusion Assay to Study Measles Virus Entry.

We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter system is comprised of two expression vectors, each encoding a segment of Renilla luciferase fused to a segment of GFP. To regain function, the two segments need to associate, which is dependent on cell-cell fusion between effector cells expressing the MeV fusion machinery and target cells expressing the corresponding MeV receptor. By measuring reconstituted luciferase activity, we can follow the kinetics of cell-cell fusion and quantify the extent of fusion. This assay lends itself to the study of the MeV fusion machinery comprised of the attachment and fusion glycoproteins, the matrix protein, and the MeV receptors. Moreover, entry inhibitors targeting attachment or fusion can be readily screened using this assay. Finally, this assay can be easily adopted to study the entry of other members of the Paramyxoviridae, as we have demonstrated for the henipaviruses.

Microscopic Analysis of Cell Fate Alteration Induced by Cell Fusion.

In mammals, differentiated cells generally do not de-differentiate nor undergo cell fate alterations. However, they can be experimentally guided toward a different lineage. Cell fusion involving two different cell types has long been used to study this process, as this method induces cell fate alterations within hours to days in a subpopulation of fused cells, as evidenced by changes in gene-expression profiles. Despite the robustness of this system, its use has been restricted by low fusion rates and difficulty in eliminating unfused populations, thereby compromising resolution. In this study, we address these limitations by isolating fused cells using antibody-conjugated beads. This approach enables the microscopic tracking of fused cells starting as early as 5 hours after fusion. By taking advantage of species-specific FISH probes, we show that a small population of fused cells resulting from the fusion of mouse ES and human B cells, expresses OCT4 from human nuclei at levels comparable to human induced pluripotent stem cells (iPSCs) as early as 25 hours after fusion. We also show that this response can vary depending on the fusion partner. Our study broadens the usage of the cell fusion system for comprehending the mechanisms underlying cell fate alterations. These findings hold promise for diverse fields, including regenerative medicine and cancer.

Tspan5 promotes the EMT process to regulate the syncytialization of trophoblast cells by activating Notch signalling.

Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. ß-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

Genetic studies of human-chimpanzee divergence using stem cell fusions.

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.

Could the Human Endogenous Retrovirus-Derived Syncytialization Inhibitor, Suppressyn, Limit Heterotypic Cell Fusion Events in the Decidua?

Proper placental development relies on tightly regulated trophoblast differentiation and interaction with maternal cells. Human endogenous retroviruses (HERVs) play an integral role in modulating cell fusion events in the trophoblast cells of the developing placenta. Syncytin-1 (ERVW-1) and its receptor, solute-linked carrier family A member 5 (SLC1A5/ASCT2), promote fusion of cytotrophoblast (CTB) cells to generate the multi-nucleated syncytiotrophoblast (STB) layer which is in direct contact with maternal blood. Another HERV-derived protein known as Suppressyn (ERVH48-1/SUPYN) is implicated in anti-fusogenic events as it shares the common receptor with ERVW-1. Here, we explore primary tissue and publicly available datasets to determine the distribution of ERVW-1, ERVH48-1 and SLC1A5 expression at the maternal-fetal interface. While SLC1A5 is broadly expressed in placental and decidual cell types, ERVW-1 and ERVH48-1 are confined to trophoblast cell types. ERVH48-1 displays higher expression levels in CTB and extravillous trophoblast, than in STB, while ERVW-1 is generally highest in STB. We have demonstrated through gene targeting studies that suppressyn has the ability to prevent ERVW-1-induced fusion events in co-culture models of trophoblast cell/maternal endometrial cell interactions. These findings suggest that differential HERV expression is vital to control fusion and anti-fusogenic events in the placenta and consequently, any imbalance or dysregulation in HERV expression may contribute to adverse pregnancy outcomes.

Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR.

The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.

Cytoplasmic Transfer Methods for Studying the Segregation of Mitochondrial DNA in Mice.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA , respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.

Interspecies cell fusion between mouse embryonic stem cell and porcine pluripotent cell.

In the area of stem cell research, fusion of somatic cells into pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell on the pluripotent state of somatic nucleus. As many other groups, we previously established a porcine pluripotent cell line at a low potential. Therefore, here, we performed experiments to investigate if the fusion with mouse ES cell could improve the pluripotent state of porcine pluripotent cell. Our data showed that resultant mouse-porcine interspecies fused cells are AP positive, and could be passaged up to 20 passages. Different degrees of increases in expression of porcine pluripotent genes proved that pig-origin gene network can be programmed by mouse ES. Further differentiation study also confirmed these fused cells' potential to form three germ layers. However, unexpectedly, we found that chromosome loss and aberrant (especially in porcine chromosomes) is severe after the cell fusion, implying that interspecies cell fusion may be not suitable to study porcine pluripotency without additional supportive conditions for genome stabilization.

Blockade of SARS-CoV-2 spike protein-mediated cell-cell fusion using COVID-19 convalescent plasma.

The recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell-cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell-cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

RNA-Seq identifies genes whose proteins are upregulated during syncytia development in murine C2C12 myoblasts and human BeWo trophoblasts.

The fusion of villous cytotrophoblasts into the multinucleated syncytiotrophoblast is critical for the essential functions of the mammalian placenta. Using RNA-Seq gene expression, quantitative protein expression, and siRNA knockdown we identified genes and their cognate proteins which are similarly upregulated in two cellular models of mammalian syncytia development (human BeWo cytotrophoblast to syncytiotrophoblast and murine C2C12 myoblast to myotube). These include DYSF, PDE4DIP, SPIRE2, NDRG1, PLEC, GPR146, HSPB8, DHCR7, and HDAC5. These findings provide avenues for further understanding of the mechanisms underlying mammalian placental syncytiotrophoblast development.

Use of cell fusion proteins to enhance adenoviral vector efficacy as an anti-cancer therapeutic.

Oncolytic viruses are designed to replicate in and kill cancer cells, and have shown tremendous promise in preclinical and clinical studies. Indeed, several oncolytic viruses are available to patients in a number of different countries around the world. However, most oncolytic viruses show a poor ability to spread throughout the tumor mass, frequently leading to only a partial response and regrowth of the tumor. One approach to improve spread of the viral effect throughout the tumor mass is to arm the oncolytic virus with a fusogenic protein. In this manner, a single infected cell can fuse with many adjacent uninfected cells, essentially amplifying the anti-tumor effects. In this review, we discuss the development and use of fusogenic proteins to enhance the efficacy of human adenovirus-based vectors for cancer therapy.

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.

B7-1 and GM-CSF enhance the anti-tumor immune effect of DC-tumor fusion vaccine in the treatment of prostate cancer.

The treatment of castration-resistant prostate cancer (CRPC) is always a difficulty in the clinic. Most patients with localized tumor eventually develop CRPC, even if hormone therapy is initially effective. Increasing evidence shows immunotherapy has special advantages compared with traditional therapy in cancer treatment. In this study, we constructed the DC-PC-3 fusion vaccine with B7-1- and GM-CSF-specific modification, and studied its ability to stimulate specific immune response and anti-tumor effect in vitro. The results showed that fusion of DC and tumor cells can improve the expression of associated antigens of DCs. DC-tumor fusion vaccine can strongly promote T cell proliferation and IFN-γ secretion and induce a significant tumor-specific cytotoxic T lymphocyte response. In addition, the B7-1/GM-CSF-modified fusion vaccine showed a more significant anti-tumor effect and greater ability to stimulate the immune response than that without specific modification in vitro. Thus, GM-CSF/B7-1-modified fusion vaccine might be used as a potential therapy strategy for prostate cancer.

Partial Sperm beta1 Integrin Subunit Deletion Proves its Involvement in Mouse Gamete Adhesion/Fusion.

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Letrozole Suppresses the Fusion of Osteoclast Precursors through Inhibition of p38-Mediated DC-STAMP Pathway.

Letrozole is a reversible nonsteroidal aromatase inhibitor that is widely used in postmenopausal breast cancer patients. It is well established that letrozole decreases bone density owing to estrogen depletion; however, few studies have reported its direct effect on bone cells in vitro. Therefore, we investigated the effect of letrozole on bone metabolism, focusing on osteoclastogenesis. Letrozole did not affect the viability, proliferation, or migration of bone marrow-derived macrophages (BMMs); however, it reduced the multinucleation of immature osteoclasts and subsequent bone resorption in vitro. Overall, letrozole inhibited the expression of dendritic cell-specific transmembrane protein (DC-STAMP), tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Among them, the reduced expression of DC-STAMP was the most prominent. However, this downregulation of DC-STAMP expression following letrozole treatment was not related to the inhibition of major osteoclastogenesis pathways, such as the nuclear factor-κB (NF-κB), c-Fos, and nuclear factor of activated T cell c1 (NFATc1) pathways, but was attributed to the inhibition of p38, which is known to reside upstream of DC-STAMP expression. Notably, the anti-osteoclastogenic effect of letrozole was abolished following treatment with the p38 activator anisomycin. Contrary to our expectations, these results strongly suggest a previously unknown anti-osteoclastogenic activity of letrozole, mediated by the downregulation of the p38/DC-STAMP pathway.

Altered Tumor Plasticity after Different Cancer Cell Fusions with MSC.

While cell fusion demonstrates an important pathway during tissue development and regeneration of distinct organs, this process can also contribute to pathophysiological phenotypes during tumor progression. Hybrid cell formation after heterofusion between cancer cells and various other cell types within the tumor microenvironment is observed in vitro and in vivo. In particular, mesenchymal stroma/stem-like cells (MSC) perform diverse levels of communication with cancer cells by exhibiting anti- and pro-tumorigenic effects. During these cellular interactions, MSC can eventually fuse with cancer cells. Thereby, the newly generated disparate hybrid populations display aneuploidy associated with chromosomal instability. Based upon a subsequent post-hybrid selection process (PHSP), fused cancer cells can undergo apoptosis/necroptosis, senescence, dormancy, or a proliferative state by acquisition of new properties. Consequently, PHSP-surviving hybrid cancer cells demonstrate altered functionalities within the tumor tissue. This is accompanied by changes in therapeutic responsiveness and a different metastatic behavior. Accordingly, enhanced tumor plasticity interferes with successful therapeutic interventions and aggravates patient prognoses. The present review article focusses on fusion of MSC with different human cancer cells, in particular breast cancer populations and resulting characteristics of various cancer hybrid cells. Moreover, some mechanisms of cancer cell fusion are discussed together with multiple PHSP pathways.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria.

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.