Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

Cellular therapy is considered a better option for the treatment of degenerative disorders. Different cell types are being used for tissue regeneration. Despite extensive research in this field, several issues remain to be addressed concerning cell transplantation. One of these issues is the survival and homing of administered cells in the injured tissue, which depends on the ability of these cells to adhere. To enhance cell adherence and survival, Rap1 GTPase was activated in mesenchymal stem cells (MSCs) as well as in cardiomyocytes (CMs) by using 8-pCPT-2\'-O-Me-cAMP, and the effect on gene expression dynamics was determined through quantitative reverse transcriptase-polymerase chain reaction analysis. Pharmacological activation of MSCs and CMs resulted in the upregulation of connexin-43 and cell adhesion genes, which increased the cell adhesion ability of MSCs and CMs, and increased the fusion of MSCs with neonatal CMs. Treating stem cells with a pharmacological agent that activates Rap1a before transplantation can enhance their fusion with CMs and increase cellular regeneration.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

BACKGROUND: Sex-specific morphogenesis occurs in Caenorhabditis elegans in the vulva of the hermaphrodite and in the male tail during the last larval stage. Temporal progression of vulva morphogenesis has been described in fine detail. However, a similar precise description of male tail morphogenesis was lacking.
RESULTS: We here describe morphogenesis of the male tail at time points matching vulva development with special focus on morphogenesis of the tail tip. Using fluorescent reporters, we follow changes in cell shapes, cell fusions, nuclear migration, modifications in the basement membrane, and formation of a new apical extracellular matrix at the end of the tail.
CONCLUSIONS: Our analysis answers two open questions about tail tip morphogenesis (TTM) by showing that one of the four tail tip cells, hyp11, remains largely separate, while the other cells fully fuse with each other and with two additional tail cells to form a ventral tail syncytium. This merger of cells begins at the apical surface early during TTM but is only completed toward the end of the process. This work provides a framework for future investigations of cell biological factors that drive male tail morphogenesis.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG\'s influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG\'s inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG\'s multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG\'s inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

The etiology of preeclampsia (PE), a complex and multifactorial condition, remains incompletely understood. DNA methylation, which is primarily regulated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, plays a vital role in early embryonic development and trophectoderm differentiation. Yet, how DNMTs modulate trophoblast fusion and PE development remains unclear. In this study, we found that the DNMTs expression was downregulated during trophoblast cells fusion. Downregulation of DNMTs was observed during the reconstruction of the denuded syncytiotrophoblast (STB) layer of placental explants. Additionally, overexpression of DNMTs inhibited trophoblast fusion. Conversely, treatment with the DNA methylation inhibitor 5-aza-CdR decreased the expression of DNMTs and promoted trophoblast fusion. A combined analysis of DNA methylation data and gene transcriptome data obtained from the primary cytotrophoblasts (CTBs) fusion process identified 104 potential methylation-regulated differentially expressed genes (MeDEGs) with upregulated expression due to DNA demethylation, including CD59, TNFAIP3, SDC1, and CDK6. The transcription regulation region (TRR) of TNFAIP3 showed a hypomethylation with induction of 5-aza-CdR, which facilitated CREB recruitment and thereby participated in regulating trophoblast fusion. More importantly, clinical correlation analysis of PE showed that the abnormal increase in DNMTs may be involved in the development of PE. This study identified placental DNA methylation-regulated genes that may contribute to PE, offering a novel perspective on the role of epigenetics in trophoblast fusion and its implication in PE development.

Understanding the mechanisms of polyploidization in cardiomyocytes is crucial for advancing strategies to stimulate myocardial regeneration. Although endoreplication has long been considered the primary source of polyploid human cardiomyocytes, recent animal work suggests the potential for cardiomyocyte fusion. Moreover, the effects of polyploidization on the genomic-transcriptomic repertoire of human cardiomyocytes have not been studied previously. We applied single-nuclei whole genome sequencing, single nuclei RNA sequencing, and multiome ATAC + gene expression (from the same nuclei) techniques to nuclei isolated from 11 healthy hearts. Utilizing post-zygotic non-inherited somatic mutations occurring during development as \"endogenous barcodes,\" to reconstruct lineage relationships of polyploid cardiomyocytes. Of 482 cardiomyocytes from multiple healthy donor hearts 75.7% can be sorted into several developmental clades marked by one or more somatic single-nucleotide variants (SNVs). At least ~10% of tetraploid cardiomyocytes contain cells from distinct clades, indicating fusion of lineally distinct cells, whereas 60% of higher-ploidy cardiomyocytes contain fused cells from distinct clades. Combined snRNA-seq and snATAC-seq revealed transcriptome and chromatin landscapes of polyploid cardiomyocytes distinct from diploid cardiomyocytes, and show some higher-ploidy cardiomyocytes with transcriptional signatures suggesting fusion between cardiomyocytes and endothelial and fibroblast cells. These observations provide the first evidence for cell and nuclear fusion of human cardiomyocytes, raising the possibility that cell fusion may contribute to developing or maintaining polyploid cardiomyocytes in the human heart.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the \"LKTG\" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.