The Fertilization Enigma: How Sperm and Egg Fuse.

Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm-egg fusion, have remained mysterious for many decades. Here, we summarize our current molecular understanding of sperm-egg interaction, focusing mainly on mammalian fertilization. Given the fundamental importance of sperm-egg fusion yet the lack of knowledge of this process in vertebrates, we discuss hallmarks and emerging themes of cell fusion by drawing from well-studied examples such as viral entry, placenta formation, and muscle development. We conclude by identifying open questions and exciting avenues for future studies in gamete fusion.

Drosophila Myoblast Fusion: Invasion and Resistance for the Ultimate Union.

Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.

SARS-CoV-2 spreads through cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

Histone deacetylase inhibitor boosts anticancer potential of fusogenic oncolytic vaccinia virus by enhancing cell-cell fusion.

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non-fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non-fusogenic VV. These functions depend strongly on cell-cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell-cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion-based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16-F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non-fusogenic VV. Moreover, TSA enhanced cell-cell fusion and FUVAC cytotoxicity in these tumor cells in a dose-dependent manner. Transcriptome analysis revealed that TSA-treated tumors showed altered expression of cellular component-related genes, which may affect fusion tolerance. In a bilateral tumor-bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non-fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell-cell fusion efficacy of FUVAC.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

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.

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell-cell fusion.

Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone-GPCR (G-protein-coupled receptor)-MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell-cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception.

Cell-cell fusions and cell-in-cell phenomena in healthy cells and cancer: Lessons from protists and invertebrates.

Herein we analyze two special routes of the multinucleated cells' formation - the fusion of mononuclear cells and the formation of cell-in-cell structures - in the healthy tissues and in tumorigenesis. There are many theories of tumorigenesis based on the phenomenon of emergence of the hybrid cancer cells. We consider the phenomena, which are rarely mentioned in those theories: namely, cellularization of syncytium or coenocytes, and the reversible or irreversible somatogamy. The latter includes the short-term and the long-term vegetative (somatic) cells' fusions in the life cycles of unicellular organisms. The somatogamy and multinuclearity have repeatedly and independently emerged in various groups of unicellular eukaryotes. These phenomena are among dominant survival and biodiversity sustaining strategies in protists and we admit that they can likely play an analogous role in cancer cells.

Verifying AXL and putative proteins as SARS-CoV-2 receptors by DnaE intein-based rapid cell-cell fusion assay.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

Establishment of a system to quantify wild-type herpes simplex virus-induced cell-cell fusion reveals a role of N-glycosylation of HSV-1 envelope glycoprotein B in cell-cell fusion.

Wild-type herpes simplex virus (HSV) strains infrequently mediate cell-cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell-cell fusion induced by wild-type HSV strains. The established system clarified that the HSV-1 envelope glycoprotein B and its N-glycosylation at asparagine at position 141 were required for efficient cell-cell fusion. This study provides a link between cell-cell fusion induced by wild-type HSV-1 and viral pathogenesis in vivo.

Long COVID as a Tauopathy: Of "Brain Fog" and "Fusogen Storms".

Long COVID, also called post-acute sequelae of SARS-CoV-2, is characterized by a multitude of lingering symptoms, including impaired cognition, that can last for many months. This symptom, often called "brain fog", affects the life quality of numerous individuals, increasing medical complications as well as healthcare expenditures. The etiopathogenesis of SARS-CoV-2-induced cognitive deficit is unclear, but the most likely cause is chronic inflammation maintained by a viral remnant thriving in select body reservoirs. These viral sanctuaries are likely comprised of fused, senescent cells, including microglia and astrocytes, that the pathogen can convert into neurotoxic phenotypes. Moreover, as the enteric nervous system contains neurons and glia, the virus likely lingers in the gastrointestinal tract as well, accounting for the intestinal symptoms of long COVID. Fusogens are proteins that can overcome the repulsive forces between cell membranes, allowing the virus to coalesce with host cells and enter the cytoplasm. In the intracellular compartment, the pathogen hijacks the actin cytoskeleton, fusing host cells with each other and engendering pathological syncytia. Cell-cell fusion enables the virus to infect the healthy neighboring cells. We surmise that syncytia formation drives cognitive impairment by facilitating the "seeding" of hyperphosphorylated Tau, documented in COVID-19. In our previous work, we hypothesized that the SARS-CoV-2 virus induces premature endothelial senescence, increasing the permeability of the intestinal and blood-brain barrier. This enables the migration of gastrointestinal tract microbes and/or their components into the host circulation, eventually reaching the brain where they may induce cognitive dysfunction. For example, translocated lipopolysaccharides or microbial DNA can induce Tau hyperphosphorylation, likely accounting for memory problems. In this perspective article, we examine the pathogenetic mechanisms and potential biomarkers of long COVID, including microbial cell-free DNA, interleukin 22, and phosphorylated Tau, as well as the beneficial effect of transcutaneous vagal nerve stimulation.

Altered Phenotypes of Breast Epithelial × Breast Cancer Hybrids after ZEB1 Knock-Out.

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness.

Mechanisms regulating myoblast fusion: A multilevel interplay.

Myoblast fusion into myotubes is one of the crucial steps of skeletal muscle development (myogenesis). The fusion is preceded by specification of a myogenic lineage (mesodermal progenitors) differentiating into myoblasts and is followed by myofiber-type specification and neuromuscular junction formation. Similarly to other processes of myogenesis, the fusion requires a very precise spatial and temporal regulation occuring both during embryonic development as well as regeneration and repair of the muscle. A plethora of genes and their products is involved in regulation of myoblast fusion and a precise multilevel interplay between them is crucial for myogenic cells to fuse. In this review, we describe both cellular events taking place during myoblast fusion (migration, adhesion, elongation, cell-cell recognition, alignment, and fusion of myoblast membranes enabling formation of myotubes) as well as recent findings on mechanisms regulating this process. Also, we present muscle disorders in humans that have been associated with defects in genes involved in regulation of myoblast fusion.

LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

Reconstitution of Fusion-Competent Human Placental Fusogen Syncytin-2.

Mammalian placenta formation requires continuous fusion of trophoblasts. Human endogenous retrovirus-derived proteins syncytin-1 and syncytin-2 mediate cell-cell fusion of placental cytotrophoblasts to form syncytiotrophoblasts in primates, which is required for normal placenta function and fetal development. Syncytins are post-translationally cleaved by the endoprotease furin into surface (SU) and transmembrane (TM) subunits for activation. Little is currently known about the molecular mechanisms of syncytin-mediated cell-cell fusion, and their functions have not been well studied in vitro. Here, we express tagged syncytin-2 in mammalian HEK293T cells and demonstrate that the tagging greatly influences the cleavage and fusogenic activity of syncytin-2. By detecting the N-terminal tagged SU, we find that it is released into the extracellular space during the fusion process. Furthermore, when N-linked glycosylation and disulfide bond formation are blocked, the cleavage and fusogenic activity of syncytin-2 are inhibited. Finally, we were able to purify functional syncytin-2 from HEK293T cells and incorporate it into proteoliposomes. These findings lay a solid foundation for interogating the molecular mechanisms of syncytin-2-mediated cell-cell fusion in vitro.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.

Aggregation of high-frequency RBD mutations of SARS-CoV-2 with three VOCs did not cause significant antigenic drift.

Variants of SARS-CoV-2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high-frequency naturally occurring amino acid changes in the receptor-binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2-overexpressing 293T cells of different species, LLC-MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high-frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high-frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

Generation of Cancer Stem/Initiating Cells by Cell-Cell Fusion.

CS/ICs have raised great expectations in cancer research and therapy, as eradication of this key cancer cell type is expected to lead to a complete cure. Unfortunately, the biology of CS/ICs is rather complex, since no common CS/IC marker has yet been identified. Certain surface markers or ALDH1 expression can be used for detection, but some studies indicated that cancer cells exhibit a certain plasticity, so CS/ICs can also arise from non-CS/ICs. Another problem is intratumoral heterogeneity, from which it can be inferred that different CS/IC subclones must be present in the tumor. Cell-cell fusion between cancer cells and normal cells, such as macrophages and stem cells, has been associated with the generation of tumor hybrids that can exhibit novel properties, such as an enhanced metastatic capacity and even CS/IC properties. Moreover, cell-cell fusion is a complex process in which parental chromosomes are mixed and randomly distributed among daughter cells, resulting in multiple, unique tumor hybrids. These, if they have CS/IC properties, may contribute to the heterogeneity of the CS/IC pool. In this review, we will discuss whether cell-cell fusion could also lead to the origin of different CS/ICs that may expand the overall CS/IC pool in a primary tumor.

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.