scholarly journals Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell–cell fusion

2012 ◽  
Vol 197 (4) ◽  
pp. 553-568 ◽  
Author(s):  
Tsukasa Oikawa ◽  
Masaaki Oyama ◽  
Hiroko Kozuka-Hata ◽  
Shunsuke Uehara ◽  
Nobuyuki Udagawa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Cell Fusion ◽  
Underlying Mechanism ◽  
Hybrid Cells ◽  
Multinucleated Cells ◽  
Phosphoinositide 3 Kinase ◽  
Inflammatory Milieu ◽  
Invadopodia Formation ◽  
Cell Cell

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src−/− osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma–osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell–cell fusion in and beyond osteoclasts.

scholarly journals Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Olivia Muriel ◽  
Laetitia Michon ◽  
Wanda Kukulski ◽  
Sophie G. Martin
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Turgor Pressure ◽  
Light And Electron Microscopy ◽  
Asymmetric Membrane ◽  
Local Reduction ◽  
Fusion Site ◽  
Actin Structure ◽  
Fusion Pores ◽  
Cell Cell

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.

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

2020 ◽  
Vol 118 (1) ◽  
pp. e2007526118
Author(s):  
Ka Man Carmen Chan ◽  
Ashley L. Arthur ◽  
Johannes Morstein ◽  
Meiyan Jin ◽  
Abrar Bhat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Cell Fusion ◽  
Conformational Changes ◽  
Binding Motif ◽  
Mechanical Pressure ◽  
Actin Nucleation ◽  
Viral Spread ◽  
Fast Proteins ◽  
Cell Cell

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.

scholarly journals Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

2021 ◽  
Author(s):  
Olivia Muriel ◽  
Laetitia Michon ◽  
Wanda Kukulski ◽  
Sophie G Martin
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Sexual Reproduction ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Turgor Pressure ◽  
Wall Material ◽  
M Cells ◽  
P Cells ◽  
Cell Cell

Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo- to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P- than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

2021 ◽  
Author(s):  
Yao Yu Yeo ◽  
David W. Buchholz ◽  
Amandine Gamble ◽  
Mason Jager ◽  
Hector C. Aguilar
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Emerging Viruses ◽  
Multinucleated Cells ◽  
Head Domain ◽  
Cell Cell

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

scholarly journals Cell–cell fusion of mesenchymal cells with distinct differentiations triggers genomic and transcriptomic remodelling toward tumour aggressiveness

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lucile Delespaul ◽  
Caroline Gélabert ◽  
Tom Lesluyes ◽  
Sophie Le Guellec ◽  
Gaëlle Pérot ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Cell Fusion ◽  
Mesenchymal Cells ◽  
Tumour Cells ◽  
Physiological Process ◽  
Hybrid Cells ◽  
Transcriptomic Profile ◽  
Transformed Fibroblasts ◽  
Histological Characteristics ◽  
Cell Cell

AbstractCell–cell fusion is a physiological process that is hijacked during oncogenesis and promotes tumour evolution. The main known impact of cell fusion is to promote the formation of metastatic hybrid cells following fusion between mobile leucocytes and proliferating tumour cells. We show here that cell fusion between immortalized myoblasts and transformed fibroblasts, through genomic instability and expression of a specific transcriptomic profile, leads to emergence of hybrid cells acquiring dissemination properties. This is associated with acquisition of clonogenic ability by fused cells. In addition, by inheriting parental properties, hybrid tumours were found to mimic the histological characteristics of a specific histotype of sarcomas: undifferentiated pleomorphic sarcomas with incomplete muscular differentiation. This finding suggests that cell fusion, as macroevolution event, favours specific sarcoma development according to the differentiation lineage of parent cells.

scholarly journals Murine Coronavirus Requires Lipid Rafts for Virus Entry and Cell-Cell Fusion but Not for Virus Release

2005 ◽  
Vol 79 (15) ◽  
pp. 9862-9871 ◽  
Author(s):  
Keum S. Choi ◽  
Hideki Aizaki ◽  
Michael M. C. Lai
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cell Fusion ◽  
Lipid Raft ◽  
Virus Entry ◽  
Buoyant Density ◽  
Spike Protein ◽  
Cholesterol Depletion ◽  
Virus Binding ◽  
Cell Cell

ABSTRACT Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl β-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4°C but shifted to lipid rafts at 37°C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.

scholarly journals A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 229 ◽  
Author(s):  
María García-Murria ◽  
Neus Expósito-Domínguez ◽  
Gerard Duart ◽  
Ismael Mingarro ◽  
Luis Martinez-Gil
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
High Throughput Screening ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Viral Proteins ◽  
Viral Fusion ◽  
Multinucleated Cells ◽  
Virus Entry Inhibitors ◽  
Cell Cell

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.

scholarly journals Myoblasts and macrophages share molecular components that contribute to cell–cell fusion

2008 ◽  
Vol 180 (5) ◽  
pp. 1005-1019 ◽  
Author(s):  
Kostandin V. Pajcini ◽  
Jason H. Pomerantz ◽  
Ozan Alkan ◽  
Regis Doyonnas ◽  
Helen M. Blau
Keyword(s):  
Cell Fusion ◽  
Cell Formation ◽  
Cell Types ◽  
Giant Cells ◽  
Nucleotide Exchange ◽  
Multinucleated Cells ◽  
Guanine Nucleotide Exchange ◽  
Distinct Cell ◽  
Molecular Components ◽  
Cell Cell

Cell–cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and β1-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.

scholarly journals Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Stefan L. Oliver ◽  
Edward Yang ◽  
Ann M. Arvin
Keyword(s):  
Varicella Zoster Virus ◽  
Cell Fusion ◽  
Cellular Responses ◽  
Melanoma Cells ◽  
Live Cell ◽  
Effective Vaccine ◽  
Viral Glycoprotein ◽  
Varicella Zoster ◽  
Multinucleated Cells ◽  
Cell Cell

ABSTRACT The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

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