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Sample records for cell fusion induced

  1. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    PubMed Central

    Yang, Po-Fu; Wang, Chih-Hung; Lee, Gwo-Bin

    2016-01-01

    Cell fusion is a critical operation for numerous biomedical applications including cell reprogramming, hybridoma formation, cancer immunotherapy, and tissue regeneration. However, unstable cell contact and random cell pairings have limited efficiency and yields when utilizing traditional methods. Furthermore, it is challenging to selectively perform cell fusion within a group of cells. This study reports a new approach called optically-induced cell fusion (OICF), which integrates cell-pairing microstructures with an optically-induced, localized electrical field. By projecting light patterns onto a photoconductive film (hydrogen-rich, amorphous silicon) coated on an indium-tin-oxide (ITO) glass while an alternating current electrical field was applied between two such ITO glass slides, “virtual” electrodes could be generated that could selectively fuse pairing cells. At 10 kHz, a 57% cell paring rate and an 87% fusion efficiency were successfully achieved at a driving voltage of 20  Vpp, suggesting that this new technology could be promising for selective cell fusion within a group of cells. PMID:26912054

  2. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    NASA Astrophysics Data System (ADS)

    Yang, Po-Fu; Wang, Chih-Hung; Lee, Gwo-Bin

    2016-02-01

    Cell fusion is a critical operation for numerous biomedical applications including cell reprogramming, hybridoma formation, cancer immunotherapy, and tissue regeneration. However, unstable cell contact and random cell pairings have limited efficiency and yields when utilizing traditional methods. Furthermore, it is challenging to selectively perform cell fusion within a group of cells. This study reports a new approach called optically-induced cell fusion (OICF), which integrates cell-pairing microstructures with an optically-induced, localized electrical field. By projecting light patterns onto a photoconductive film (hydrogen-rich, amorphous silicon) coated on an indium-tin-oxide (ITO) glass while an alternating current electrical field was applied between two such ITO glass slides, “virtual” electrodes could be generated that could selectively fuse pairing cells. At 10 kHz, a 57% cell paring rate and an 87% fusion efficiency were successfully achieved at a driving voltage of 20  Vpp, suggesting that this new technology could be promising for selective cell fusion within a group of cells.

  3. Femtosecond laser-induced fusion of nonadherent cells and two-cell porcine embryos

    NASA Astrophysics Data System (ADS)

    Kuetemeyer, Kai; Lucas-Hahn, Andrea; Petersen, Bjoern; Niemann, Heiner; Heisterkamp, Alexander

    2011-08-01

    Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm2 (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm2 (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.

  4. Laser-induced fusion of human embryonic stem cells with optical tweezers

    SciTech Connect

    Chen Shuxun; Wang Xiaolin; Sun Dong; Cheng Jinping; Han Cheng, Shuk; Kong, Chi-Wing; Li, Ronald A.

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  5. Laser-induced fusion of human embryonic stem cells with optical tweezers

    NASA Astrophysics Data System (ADS)

    Chen, Shuxun; Cheng, Jinping; Kong, Chi-Wing; Wang, Xiaolin; Han Cheng, Shuk; Li, Ronald A.; Sun, Dong

    2013-07-01

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  6. Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response▿ †

    PubMed Central

    Herschke, F.; Plumet, S.; Duhen, T.; Azocar, O.; Druelle, J.; Laine, D.; Wild, T. F.; Rabourdin-Combe, C.; Gerlier, D.; Valentin, H.

    2007-01-01

    Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-β) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-β response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-α/β production, an amplified IFN-β response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-β gene deficiencies were trans complemented to induce IFN-β production. Production of IFN-β and IFN-α was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-α/β. The amplification of IFN-β production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-α/β production in infected cells, and this indicates that MGC contribute to the antiviral immune response. PMID:17898060

  7. Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion.

    PubMed

    Atanasiu, Doina; Saw, Wan Ting; Eisenberg, Roselyn J; Cohen, Gary H

    2016-09-14

    Receptor dependent HSV-induced fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers transformation of the pre-fusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor dependent cell-cell fusion we took advantage of our discovery that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH2/gL2, thereby enhancing their activity. We also found that deregulated forms of gD1 and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

  8. [Molecular Mechanism of Glycoprotein-induced Cell-Cell Fusion of Herpesviruses].

    PubMed

    Feng, Daishen; Jia, Renyong

    2016-01-01

    Herpesviridae is a large family comprising linear, double-stranded DNA viruses. Herpesviridae contains three subfamilies: α-, β- and γ-herpesviruses. The glycoproteins gB, gH and gL of each subfamily form the "core fusion function" in cell-cell fusion. Other herpesviruses also need additional glycoproteins to promote fusion, such as gD of the Herpes simplex virus, gp42 of the Epstein-Barr virus, and gO or UL128-131 of the Human cytomegalovirus. In contrast, glycoproteins gM or gM/gN of herpesvirus inhibit fusion. We describe the molecular mechanisms of glycoprotein-induced fusion and entry of herpesviruses. It will be helpful to further study the pathogenic mechanism of herpesvirus.

  9. Polyethylene glycol-induced fusion of two-cell mouse embryo blastomeres

    SciTech Connect

    Spindle, A.

    1981-01-01

    Polyethylene glycol (PEG) was found to be an effective fusion-inducing agent for early mouse embryo blastomeres. A brief exposure of zona-intact 2-cell embryos to 40% PEG induced fusion of blastomeres in > 80% of embryos, and the treatment did not interfere with subsequent development of embryos to the blastocyst stage.

  10. Genetic studies of cell fusion induced by herpes simplex virus type 1

    SciTech Connect

    Read, G.S.; Person, S.; Keller, P.M.

    1980-07-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.

  11. Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

    PubMed Central

    Saw, Wan Ting; Eisenberg, Roselyn J.; Cohen, Gary H.

    2016-01-01

    ABSTRACT Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when

  12. Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell.

    PubMed

    Markosyan, Ruben M; Kielian, Margaret; Cohen, Fredric S

    2007-10-01

    Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.

  13. Lipopolysaccharide-induced multinuclear cells: Increased internalization of polystyrene beads and possible signals for cell fusion

    SciTech Connect

    Nakanishi-Matsui, Mayumi Yano, Shio; Futai, Masamitsu

    2013-11-01

    Highlights: •LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. •Large beads are internalized by cells actively fusing to become multinuclear. •The multinuclear cell formation is inhibited by anti-inflammatory cytokine, IL10. •Signal transduction for cell fusion is different from that for inflammation. -- Abstract: A murine macrophage-derived line, RAW264.7, becomes multinuclear on stimulation with lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria. These multinuclear cells internalized more polystyrene beads than mononuclear cells or osteoclasts (Nakanishi-Matsui, M., Yano, S., Matsumoto, N., and Futai, M., 2012). In this study, we analyzed the time courses of cell fusion in the presence of large beads. They were internalized into cells actively fusing to become multinuclear. However, the multinuclear cells once formed showed only low phagocytosis activity. These results suggest that formation of the multinuclear cells and bead internalization took place simultaneously. The formation of multinuclear cells was blocked by inhibitors for phosphoinositide 3-kinase, phospholipase C, calcineurin, and c-Jun N-terminal kinase. In addition, interleukin 6 and 10 also exhibited inhibitory effects. These signaling molecules and cytokines may play a crucial role in the LPS-induced multinuclear cell formation.

  14. Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion.

    PubMed Central

    Lang, R D; Wickenden, C; Wynne, J; Lucy, J A

    1984-01-01

    Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide

  15. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion.

    PubMed

    Schmid, E; Zurbriggen, A; Gassen, U; Rima, B; ter Meulen, V; Schneider-Schaulies, J

    2000-08-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.

  16. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

  17. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    SciTech Connect

    Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  18. Lipopolysaccharide-induced multinuclear cells: increased internalization of polystyrene beads and possible signals for cell fusion.

    PubMed

    Nakanishi-Matsui, Mayumi; Yano, Shio; Futai, Masamitsu

    2013-11-01

    A murine macrophage-derived line, RAW264.7, becomes multinuclear on stimulation with lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria. These multinuclear cells internalized more polystyrene beads than mononuclear cells or osteoclasts (Nakanishi-Matsui, M., Yano, S., Matsumoto, N., and Futai, M., 2012). In this study, we analyzed the time courses of cell fusion in the presence of large beads. They were internalized into cells actively fusing to become multinuclear. However, the multinuclear cells once formed showed only low phagocytosis activity. These results suggest that formation of the multinuclear cells and bead internalization took place simultaneously. The formation of multinuclear cells was blocked by inhibitors for phosphoinositide 3-kinase, phospholipase C, calcineurin, and c-Jun N-terminal kinase. In addition, interleukin 6 and 10 also exhibited inhibitory effects. These signaling molecules and cytokines may play a crucial role in the LPS-induced multinuclear cell formation. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Evidence of cell fusion in carcinogen-induced mice gastric carcinoma.

    PubMed

    Yan, Yongjia; Hsu, Yiling; He, Xianghui; Lu, Ning; Wei, Wei; Zhang, Zhixiang; Zhu, Liwei

    2015-07-01

    The role of bone marrow-derived cells in gastric cancer formation was not fully understood. In this study, bone marrow from female green fluorescent protein transgenic mice was transplanted into male wild-type mice to generate sex-mismatched chimeric mice. The chimeric mice were treated with carcinogen to induce gastric cancer. At time of sacrifice, 18.2 % (2/11) of mice showed severe dysplasia and 25 % (3/12) of mice successfully induced with cancer. Fluorescence in situ hybridization results showed that bone marrow-derived cells participated in renewal of gastric mucosa and cell fusion was observed in both precancerous lesions and adenocarcinoma, but no sign of fusion was observed in squamous cell carcinoma. Our findings suggest that bone marrow-derived cells participate in renewal of gastric mucosa during chronic damage and might have acquired the phenotype of gastric epithelial cells through cell fusion. Fusion between gastric epithelial cells and bone marrow-derived cells was involved in increased carcinogenesis.

  20. Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion.

    PubMed

    Yang, Yihong; Zhang, Yan; Li, Wen-Jun; Jiang, Yuxiang; Zhu, Zhiwen; Hu, Huifang; Li, Wei; Wu, Jia-Wei; Wang, Zhi-Xin; Dong, Meng-Qiu; Huang, Shanjin; Ou, Guangshuo

    2017-04-10

    Cell-cell fusion generally requires cellular fusogenic proteins and actin-propelled membrane protrusions. However, the molecular connections between fusogens and the actin cytoskeleton remain unclear. Here, we show that the Caenorhabditis elegans fusogen EFF-1 and F-actin are enriched at the cortex of the post-embryonic fusing cells, and conditional mutations of WASP and Arp2/3 delay cell-cell fusion by impairing EFF-1 localization. Our affinity purification and mass spectrometry analyses determined that an actin-binding protein, spectraplakin/VAB-10A, binds to EFF-1. VAB-10A promotes cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Conversely, EFF-1 enhanced the F-actin bundling activity of VAB-10A in vitro, and actin dynamics in the cortex were reduced in eff-1 or vab-10a mutants. Thus, cell-cell fusion is promoted by a positive feedback loop in which actin filaments that are crosslinked by spectraplakin to recruit fusogens to fusion sites are reinforced via fusogens, thereby increasing the probability of further fusogen accumulation to form fusion synapses. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Sodium butyrate induces DRP1-mediated mitochondrial fusion and apoptosis in human colorectal cancer cells.

    PubMed

    Tailor, Dhanir; Hahm, Eun-Ryeong; Kale, Raosaheb K; Singh, Shivendra V; Singh, Rana P

    2014-05-01

    Sodium butyrate (NaBt) is the byproduct of anaerobic microbial fermentation inside the gastro-intestinal tract that could reach up to 20mM, and has been shown to inhibit the growth of various cancers. Herein, we evaluated its effect on mitochondrial fusion and associated induction of apoptosis in colorectal cancer cells (CRC). NaBt treatment at physiological (1-5mM) concentrations for 12 and 24h decreased the cell viability and induced G2-M phase cell cycle arrest in HCT116 (12h) and SW480 human CRC cells. This cell cycle arrest was associated with mitochondria-mediated apoptosis accompanied by a decrease in survivin and Bcl-2 expression, and generation of reactive oxygen species (ROS). Furthermore, NaBt treatment resulted in a significant decrease in the mitochondrial mass which is an indicator of mitochondrial fusion. Level of dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission and fusion where its up-regulation correlates with fission, was found to be decreased in CRC cells. Further, at early treatment time, DRP1 down-regulation was noticed in mitochondria which later became drastically reduced in both mitochondria as well as cytosol. DRP1 is activated by cyclin B1-CDK1 complex by its ser616 phosphorylation in which both cyclin B1-CDK1 complex and phospho-DRP1 (ser616) were strongly reduced by NaBt treatment. DRP1 was observed to be regulated by apoptosis as pan-caspase inhibitor showing rescue from NaBt-induced apoptosis also caused the reversal of DRP1 to the normal level as in control proliferating cells. Together, these findings suggest that NaBt can modulate mitochondrial fission and fusion by regulating the level of DRP1 and induce cell cycle arrest and apoptosis in human CRC cells.

  2. Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

    PubMed Central

    Roy, Nathan H.; Chan, Jany; Lambelé, Marie

    2013-01-01

    HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402

  3. CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells

    PubMed Central

    El-Mesery, M; Trebing, J; Schäfer, V; Weisenberger, D; Siegmund, D; Wajant, H

    2013-01-01

    Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction. PMID:24232092

  4. Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol

    PubMed Central

    Quirk, Susan J.; Ahkong, Quet Fah; Botham, Gaynor M.; Vos, Jan; Lucy, Jack A.

    1978-01-01

    1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca2+ used, at least 80% of the cells fused after 30min at 37°C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in `ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca2+, although some cells fused when no exogenous Ca2+ was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of `ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1–2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca2+ into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed. ImagesPLATE 4PLATE 1PLATE 2PLATE 3 PMID:728105

  5. Unraveling a Three-Step Spatiotemporal Mechanism of Triggering of Receptor-Induced Nipah Virus Fusion and Cell Entry

    PubMed Central

    Liu, Qian; Stone, Jacquelyn A.; Bradel-Tretheway, Birgit; Dabundo, Jeffrey; Benavides Montano, Javier A.; Santos-Montanez, Jennifer; Biering, Scott B.; Nicola, Anthony V.; Iorio, Ronald M.; Lu, Xiaonan; Aguilar, Hector C.

    2013-01-01

    Membrane fusion is essential for entry of the biomedically-important paramyxoviruses into their host cells (viral-cell fusion), and for syncytia formation (cell-cell fusion), often induced by paramyxoviral infections [e.g. those of the deadly Nipah virus (NiV)]. For most paramyxoviruses, membrane fusion requires two viral glycoproteins. Upon receptor binding, the attachment glycoprotein (HN/H/G) triggers the fusion glycoprotein (F) to undergo conformational changes that merge viral and/or cell membranes. However, a significant knowledge gap remains on how HN/H/G couples cell receptor binding to F-triggering. Via interdisciplinary approaches we report the first comprehensive mechanism of NiV membrane fusion triggering, involving three spatiotemporally sequential cell receptor-induced conformational steps in NiV-G: two in the head and one in the stalk. Interestingly, a headless NiV-G mutant was able to trigger NiV-F, and the two head conformational steps were required for the exposure of the stalk domain. Moreover, the headless NiV-G prematurely triggered NiV-F on virions, indicating that the NiV-G head prevents premature triggering of NiV-F on virions by concealing a F-triggering stalk domain until the correct time and place: receptor-binding. Based on these and recent paramyxovirus findings, we present a comprehensive and fundamentally conserved mechanistic model of paramyxovirus membrane fusion triggering and cell entry. PMID:24278018

  6. Human Perivascular Stem Cell-Based Bone Graft Substitute Induces Rat Spinal Fusion

    PubMed Central

    Chung, Choon G.; James, Aaron W.; Asatrian, Greg; Chang, Le; Nguyen, Alan; Le, Khoi; Bayani, Georgina; Lee, Robert; Stoker, David; Zhang, Xinli

    2014-01-01

    Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair. PMID:25154782

  7. Human perivascular stem cell-based bone graft substitute induces rat spinal fusion.

    PubMed

    Chung, Choon G; James, Aaron W; Asatrian, Greg; Chang, Le; Nguyen, Alan; Le, Khoi; Bayani, Georgina; Lee, Robert; Stoker, David; Zhang, Xinli; Ting, Kang; Péault, Bruno; Soo, Chia

    2014-10-01

    Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair. ©AlphaMed Press.

  8. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

    PubMed Central

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J.; Butler, Mark S.; Edwards, David J.; Fry, Scott R.; Bermingham, Imogen M.; Cooper, Matthew A.; Young, Paul R.

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  9. Antigen-specific CD8{sup +} T cells induced by the ubiquitin fusion degradation pathway

    SciTech Connect

    Imai, Takashi; Duan Xuefeng; Hisaeda, Hajime; Himeno, Kunisuke

    2008-01-25

    We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8{sup +} T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8{sup +} T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8{sup +} T cells.

  10. Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

    PubMed Central

    Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R.; Hannah, Brian P.; Matsuda, Zene; Whitbeck, J. Charles; Cohen, Gary H.

    2013-01-01

    Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion. PMID:23946457

  11. Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

    PubMed

    Ugarte, Giorgia D; Vargas, Macarena F; Medina, Matías A; León, Pablo; Necuñir, David; Elorza, Alvaro A; Gutiérrez, Soraya E; Moon, Randall T; Loyola, Alejandra; De Ferrari, Giancarlo V

    2015-10-08

    Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia.

  12. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

  13. Ca2+ -regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells.

    PubMed

    Han, Wei-Qing; Chen, Wen-Dong; Zhang, Ke; Liu, Jian-Jun; Wu, Yong-Jie; Gao, Ping-Jin

    2016-04-01

    It has been reported that intracellular Ca2+ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca2+ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca2+ content, an effect that was inhibited by the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca2+ -regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.

  14. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    SciTech Connect

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  15. Transplantation of neuronal cells induced from human mesenchymal stem cells improves neurological functions after stroke without cell fusion.

    PubMed

    Xu, Haiyan; Miki, Kazunori; Ishibashi, Satoru; Inoue, Jun; Sun, Liyuan; Endo, Shu; Sekiya, Ichiro; Muneta, Takeshi; Inazawa, Joji; Dezawa, Mari; Mizusawa, Hidehiro

    2010-12-01

    The options for treating stroke are limited, but stem cells hold promise as a therapy because of their multipotency. Neuronal cells derived from mesenchymal stem cells (MSC) were reported to have more therapeutic effect than MSCs. For elucidating the therapeutic mechanism of neuronal cells, here we generated a model of focal cerebral infarction by performing left common carotid artery occlusion in adult gerbils. We transfected human trabecular bone-derived MSCs (hMSCs) with the Notch intracellular domain to induce their differentiation into neuronal cells (hN-MSCs). These cells were stereotaxically transplanted into the local ischemic hemisphere 4 days after the occlusion. Behavioral analyses were conducted 28 days after transplantation, and then fluorescence in situ hybridization (FISH) and a histological evaluation were performed. Histologically, transplanted cells were distributed around the periinfarct region, and approximately 8.5% and 4.2% of hN-MSCs and hMSCs survived, respectively; 53.2% ± 9.6% of hN-MSCs were microtubule-associated protein 2(+) (MAP-2(+) ) and extended neurites, whereas only 0.9% ± 0.3% of hMSCs were MAP-2(+) . In FISH, human nucleus-specific signals were detected in both hN-MSCs and hMSCs grafted brains, but no transplanted cell had a merged gerbil-specific nuclear signals. hN-MSC-transplanted animals showed significantly better recovery than animals given control vehicle in the T-maze, bilateral asymmetry, and open field tests. These findings suggested that hN-MSCs have greater therapeutic potential than hMSCs for stroke and that cell fusion does not primarily contribute to the therapeutic mechanism of MSC transplantation. Copyright © 2010 Wiley-Liss, Inc.

  16. Monoclonal antibody production by receptor-mediated electrically induced cell fusion

    NASA Astrophysics Data System (ADS)

    Lo, Mathew M. S.; Tsong, Tian Yow; Conrad, Mary K.; Strittmatter, Stephen M.; Hester, Lynda D.; Snyder, Solomon H.

    1984-08-01

    Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance1-3, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination4-9, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

  17. Regulation of cell-cell fusion by nanotopography

    PubMed Central

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-01-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions. PMID:27615159

  18. Regulation of cell-cell fusion by nanotopography

    NASA Astrophysics Data System (ADS)

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-09-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.

  19. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  20. Cell fusion and nuclear fusion in plants.

    PubMed

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Laser-induced semicold fusion

    SciTech Connect

    Steinert, C. )

    1990-01-01

    The large high-energy lasers required for inertial fusion are at present beyond state-of-the-art, and there are other problems (instability of the fuel target, suprathermal electrons, etc.) as well. Therefore, it is hoped that the energy requirement for inertial fusion can be reduced with the help of cold fusion, which takes place within the electrode material confining the fuel (avoiding instability problems). With the semicold fusion cell, laser energy is transferred into the hot part of the fuel, which is confined within the cathode in a cavity, and credit is taken from fast projectiles (tritium) stemming from the (t,p) branch of cold fusion in the cold metal lattice. The latter is the key to the model of a dynamic process for potential growth between the cold electrode and the hot confined fuel in the semicold fusion cell.

  2. Mechanical tension drives cell membrane fusion.

    PubMed

    Kim, Ji Hoon; Ren, Yixin; Ng, Win Pin; Li, Shuo; Son, Sungmin; Kee, Yee-Seir; Zhang, Shiliang; Zhang, Guofeng; Fletcher, Daniel A; Robinson, Douglas N; Chen, Elizabeth H

    2015-03-09

    Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Accumulation of a fusion protein containing 2S albumin induces novel vesicles in vegetative cells of Arabidopsis.

    PubMed

    Hayashi, M; Toriyama, K; Kondo, M; Hara-Nishimura, I; Nishimura, M

    1999-03-01

    We have previously reported that precursor-accumulating (PAC) vesicles found exclusively in developing seeds are involved in a transport of seed storage proteins, such as 2S albumin, from the endoplasmic reticulum to protein-storage vacuoles. Here, we constructed chimeric genes that encode fusion proteins consisting of both various lengths of polypeptides derived from pumpkin 2S albumin and a selectable marker enzyme, phosphinothricin acetyltransferase. The chimeric genes were expressed in transgenic Arabidopsis in order to investigate the mechanism of the PAC vesicle formation. A fusion protein expressed by one of the chimeric genes is accumulated as a proprotein-precursor form, and localized in novel vesicles of vegetative cells. The vesicles show distinct features that well much to the PAC vesicles. Despite of the accumulation of the fusion protein, the transgenic Arabidopsis is still sensitive to phosphinothricin. Phosphinothricin acetyltransferase contained in the fusion protein is obviously compartmentalized in the PAC-like vesicles that do not permit the detoxification of this herbicide. These results indicate that the PAC-like vesicle can be induced in vegetative cells by the ectopic expression of the protein that is destined to be compartmentalized into the PAC vesicles.

  4. Dihydrotestosterone stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells and induces cranial suture fusion.

    PubMed

    Lin, Ines C; Slemp, Alison E; Hwang, Catherine; Sena-Esteves, Miguel; Nah, Hyun-Duck; Kirschner, Richard E

    2007-10-01

    The higher prevalence of metopic and sagittal suture synostosis in male infants suggests a role for androgens in early craniofacial development. These experiments characterize the influence of androgen stimulation on growth and differentiation of fetal dural and calvarial bone cells and on cranial suture fusion. Primary murine fetal (E18) dural cells and calvarial osteoblasts were isolated and cultured. Cells were treated for 48 hours with 5alpha-dihydrotestosterone (0 to 1000 nM). Cell proliferation was examined by nonradioactive proliferation assay; mRNA expression of alkaline phosphatase, transforming growth factor (TGF)-beta1, and the bone matrix proteins osteopontin, osteocalcin, and type 1 collagen was determined by reverse-transcriptase polymerase chain reaction. In separate experiments, intact fetal calvariae were grown in tissue culture with 10 nM 5alpha-dihydrotestosterone for 7 and 14 days and then examined histologically. Androgen stimulation at 5 nM increased proliferation of fetal dural cells by 46.0 percent and of fetal calvarial osteoblasts by 20.5 percent. Dural expression of osteopontin, osteocalcin, and type 1 collagen was enhanced by 5alpha-dihydrotestosterone, as was that of TGF-beta1 and alkaline phosphatase. Androgen stimulation increased calvarial osteoblast expression of alkaline phosphatase and TGF-beta1 but induced little change in expression of osteocalcin, osteopontin, and type 1 collagen. In tissue culture, 5alpha-dihydrotestosterone stimulated osteoid formation and fusion of sagittal sutures. Androgen stimulation of dural cells and osteoblasts isolated from fetal calvaria promotes cell proliferation and osteoblastic differentiation and can induce cranial suture fusion. These results suggest that sex steroid hormone signaling may stimulate sutural osteogenesis by means of osteodifferentiation of dural cells, thus explaining the male prevalence of nonsyndromic craniosynostosis.

  5. Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells.

    PubMed

    Zhou, Jing; Hu, Shuai-Er; Tan, Shi-Hao; Cao, Ruoxi; Chen, Yiyang; Xia, Dajing; Zhu, Xinqiang; Yang, Xing-Fen; Ong, Choon-Nam; Shen, Han-Ming

    2012-03-01

    Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.

  6. Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle

    PubMed Central

    Jones, Gareth J.; Steinbach, Sabine; Clifford, Derek; Baldwin, Susan L.; Ireton, Gregory C.; Coler, Rhea N.; Reed, Steven G.; Vordermeier, H. Martin

    2013-01-01

    In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from M. bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1β and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB. PMID:24012566

  7. Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

    PubMed Central

    McDade, Joel R.; Michele, Daniel E.

    2014-01-01

    Mutations in the dysferlin gene resulting in dysferlin-deficiency lead to limb-girdle muscular dystrophy 2B and Myoshi myopathy in humans. Dysferlin has been proposed as a critical regulator of vesicle-mediated membrane resealing in muscle fibers, and localizes to muscle fiber wounds following sarcolemma damage. Studies in fibroblasts and urchin eggs suggest that trafficking and fusion of intracellular vesicles with the plasma membrane during resealing requires the intracellular cytoskeleton. However, the contribution of dysferlin-containing vesicles to resealing in muscle and the role of the cytoskeleton in regulating dysferlin-containing vesicle biology is unclear. Here, we use live-cell imaging to examine the behavior of dysferlin-containing vesicles following cellular wounding in muscle cells and examine the role of microtubules and kinesin in dysferlin-containing vesicle behavior following wounding. Our data indicate that dysferlin-containing vesicles move along microtubules via the kinesin motor KIF5B in muscle cells. Membrane wounding induces dysferlin-containing vesicle–vesicle fusion and the formation of extremely large cytoplasmic vesicles, and this response depends on both microtubules and functional KIF5B. In non-muscle cell types, lysosomes are critical mediators of membrane resealing, and our data indicate that dysferlin-containing vesicles are capable of fusing with lysosomes following wounding which may contribute to formation of large wound sealing vesicles in muscle cells. Overall, our data provide mechanistic evidence that microtubule-based transport of dysferlin-containing vesicles may be critical for resealing, and highlight a critical role for dysferlin-containing vesicle–vesicle and vesicle–organelle fusion in response to wounding in muscle cells. PMID:24203699

  8. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells.

    PubMed

    Bustos, Rodrigo I; Jensen, Erik L; Ruiz, Lina M; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A

    2013-08-02

    Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response.

    PubMed

    Kim, Sang-Hoon; Hur, Yu Jin; Lee, Suk Jun; Kim, Sang Joon; Park, Chung-Gyu; Oh, Yu-Koung; Jung, Woon-Won; Seo, Jong Bok; Nam, Myung Hee; Choi, Inho; Chun, Taehoon

    2011-04-01

    Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

  10. Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle.

    PubMed

    Jones, Gareth J; Steinbach, Sabine; Clifford, Derek; Baldwin, Susan L; Ireton, Gregory C; Coler, Rhea N; Reed, Steven G; Vordermeier, H Martin

    2013-10-25

    In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1β and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

    SciTech Connect

    Shen Jianying; Hisaeda, Hajime; Chou Bin; Yu Qingsheng; Tu Liping; Himeno, Kunisuke

    2008-01-25

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8{sup +} T cells. In this study, we exploited UPS to induce CD8{sup +} T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-{gamma}-producing CD8{sup +} T cells compared with those from the latter group of mice.

  12. A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct gammadelta and alphabeta T cell responses in primates.

    PubMed

    Cendron, Delphine; Ingoure, Sophie; Martino, Angelo; Casetti, Rita; Horand, Françoise; Romagné, François; Sicard, Hélène; Fournié, Jean-Jacques; Poccia, Fabrizio

    2007-02-01

    Phosphoantigens are mycobacterial non-peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for gammadelta T cell responses to mycobacteria. Thus here, the immunogenicity of 6-kDa early secretory antigenic target-mycolyl transferase complex antigen 85B (ESAT-6-Ag85B) (H-1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime-boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL-2, IL-6, IFN-gamma, TNF-alpha), it further anergized blood gammadelta T lymphocytes selectively. By contrast, the hybrid H-1 induced only memory alphabeta T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN-gamma-secreting cells (+ 40 cells/million PBMC, equally specific for ESAT-6 and for Ag85B). Hence, in macaques, a prime-boost with the H-1/phosphoantigen subunit combination induces two waves of immune responses, successively by gammadelta T and alphabeta T lymphocytes.

  13. Adenoviral Vectors Armed with Cell Fusion-Inducing Proteins as Anti-Cancer Agents

    PubMed Central

    Del Papa, Joshua; Parks, Robin J.

    2017-01-01

    Cancer is a devastating disease that affects millions of patients every year, and causes an enormous economic burden on the health care system and emotional burden on affected families. The first line of defense against solid tumors is usually extraction of the tumor, when possible, by surgical methods. In cases where solid tumors can not be safely removed, chemotherapy is often the first line of treatment. As metastatic cancers often become vigorously resistant to treatments, the development of novel, more potent and selective anti-cancer strategies is of great importance. Adenovirus (Ad) is the most commonly used virus in cancer clinical trials, however, regardless of the nature of the Ad-based therapeutic, complete responses to treatment remain rare. A number of pre-clinical studies have shown that, for all vector systems, viral spread throughout the tumor mass can be a major limiting factor for complete tumor elimination. By expressing exogenous cell-fusion proteins, many groups have shown improved spread of Ad-based vectors. This review summarizes the research done to examine the potency of Ad vectors expressing fusogenic proteins as anti-cancer therapeutics. PMID:28106842

  14. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    SciTech Connect

    Ohnuma, Kei; Uchiyama, Masahiko; Hatano, Ryo; Takasawa, Wataru; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  15. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

    SciTech Connect

    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A.

    2013-08-02

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

  16. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

  17. Virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins VP4 and VP7.

    PubMed Central

    Gilbert, J M; Greenberg, H B

    1997-01-01

    We recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbert, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582-5591, 1995). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodoptera frugiperda Sf-9 cells from recombinant baculoviruses expressing the four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activity was dependent on trypsinization of VP4, and the strain-specific phenotype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show that intact rotavirus and VLPs induce syncytia with cells that are permissive to rotavirus infection whereas nonpermissive cells are refractory to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involved in viral infection and specifically the events related to viral entry into the cell. Our results also demonstrate that neither viral replication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are required for fusion and that both VP4 and VP7 are essential. The combination of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells. PMID:9151849

  18. TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models.

    PubMed

    McCormick, Alison A; Corbo, Tina A; Wykoff-Clary, Sherri; Nguyen, Long V; Smith, Mark L; Palmer, Kenneth E; Pogue, Gregory P

    2006-09-29

    Fusion of peptides to viral carriers has proven an effective method for improving cellular immunity. In this study we explore the ability of a plant virus, Tobacco mosaic virus (TMV), to stimulate cellular immunity by interacting directly with immune cells. Fluorescently labeled TMV was incubated in vitro with murine spleen or lymph node cells, and near quantitative labeling of lymphocytes was achieved after 2 h, which persisted for up to 48 h. Direct TMV uptake and upregulation of the CD86 activation marker was measured in nearly all dendritic cells (DCs) by flow cytometry. To demonstrate that TMV can also provide functional antigen delivery and immune stimulation in vivo, two well-characterized T-cell epitopes that provide protection against tumor challenge in mice were fused to TMV coat protein by genetic manipulation, or by chemical conjugation. Vaccination of C57BL/6 mice elicited measurable cellular responses by interferon gamma (IFN gamma) ELISpot and resulted in significantly improved protection from tumor challenge in both the EG.7-Ova and B16 melanoma models. From these results we conclude that TMV was an effective antigen carrier for inducing cellular immune responses to less than 1 microg of peptide.

  19. A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

    SciTech Connect

    Schnabl, Bernd Brandl, Katharina; Fink, Marina; Gross, Philipp; Taura, Kojiro; Gaebele, Erwin; Hellerbrand, Claus; Falk, Werner

    2008-10-17

    Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NF{kappa}B and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.

  20. Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

    PubMed Central

    Kim, In-Joong; Chouljenko, Vladimir N.; Walker, Jason D.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread. PMID:23678175

  1. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

    PubMed Central

    Daniels-Wells, Tracy R.; Helguera, Gustavo; Rodríguez, José A.; Leoh, Lai Sum; Erb, Michael A.; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L.

    2012-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin. PMID:23085102

  2. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

    PubMed

    Daniels-Wells, Tracy R; Helguera, Gustavo; Rodríguez, José A; Leoh, Lai Sum; Erb, Michael A; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L

    2013-02-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

  3. B cells “transduced” with TAT-fusion proteins can induce tolerance and protect mice from diabetes and EAE

    PubMed Central

    Su, Yan; Zhang, Ai-Hong; Li, Xin; Owusu-Boaitey, Nana; Skupsky, Jonathan; Scott, David W.

    2011-01-01

    Antigen-immunoglobulin fusion protein expressing B cells have been shown as excellent tolerogenic antigen-presenting cells in multiple disease models. Using efficient protein transduction by fusion with a HIV TAT protein transduction domain, we herein tested the TAT-fusion protein transduced B cells for their effects in antigen-specific tolerance induction in two animal models, experimental autoimmune encephalitis (EAE) and type 1 diabetes. We demonstrated that transfer of TAT-MOG35-55 (myelin oligodendrocyte glycoprotein)-Ig ‘transduced B cells’ 10 days after EAE induction significantly protected mice from disease. Similarly, the onset of was delayed when NOD mice received insulin specific TAT-B9-23- B cells. Surprisingly, no protection against EAE was observed in a prophylactic protocol when transduced B cells were given before disease induction. Moreover, TAT-ovalbumin transduced cells were tolerogenic in primed but not naïve mice. Our results suggest that TAT-fusion protein transduced B cells were tolerogenic in antigen primed recipients, a condition clinically relevant to autoimmune diseases. PMID:21546313

  4. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    PubMed

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

  5. Membrane Fusion Induced by Small Molecules and Ions

    PubMed Central

    Mondal Roy, Sutapa; Sarkar, Munna

    2011-01-01

    Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganization of big molecules like proteins/peptides, supplies the required driving force to overcome the energy barrier of the different intermediate steps. Small molecules/ions do not share this advantage. Hence fusion induced by small molecules/ions is expected to be different from that induced by proteins/peptides. Although several reviews exist on membrane fusion, no recent review is devoted solely to small moleculs/ions induced membrane fusion. Here we intend to present, how a variety of small molecules/ions act as independent fusogens. The detailed mechanism of some are well understood but for many it is still an unanswered question. Clearer understanding of how a particular small molecule can control fusion will open up a vista to use these moleucles instead of proteins/peptides to induce fusion both in vivo and in vitro fusion processes. PMID:21660306

  6. Novel glycopolypeptide synthesis induced by gametic cell fusion in Chlamydomonas reinhardtii

    PubMed Central

    1978-01-01

    Within the first hour of zygote maturation, Chlamydomonas reinhardtii cells stop synthesizing certain polypeptides that characterize the vegetative and gametic stages of the life cycle and initiate the synthesis of novel, zygote-specific polypeptides. At least six of these polypeptides are secreted into the medium, and fine-structural studies indicate that they represent components of the cell wall that is synthesized and secreted early in zygote development. We conclude that a new program of protein synthesis, and possibly also gene transcription, is initiated shortly after gametic cells fuse, a program that appears highly suited to cell-differentiation studies. PMID:659511

  7. HIV-envelope-dependent cell-cell fusion: quantitative studies.

    PubMed

    Huerta, Leonor; López-Balderas, Nayali; Rivera-Toledo, Evelyn; Sandoval, Guadalupe; Gómez-Icazbalceta, Guillermo; Villarreal, Carlos; Lamoyi, Edmundo; Larralde, Carlos

    2009-08-11

    Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17-23; López-Balderas et al., 2007, Virus Res. 123, 138-146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env-mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env-mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

  8. Rabies Virus-Induced Membrane Fusion Pathway

    PubMed Central

    Gaudin, Yves

    2000-01-01

    Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers. PMID:10931871

  9. Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells

    PubMed Central

    Gong, Jianlin; Avigan, David; Chen, Dongshu; Wu, Zekui; Koido, Shigeo; Kashiwaba, Masahiro; Kufe, Donald

    2000-01-01

    We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors. PMID:10688917

  10. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion.

    PubMed

    Paquette, Stéphane G; Banner, David; Chi, Le Thi Bao; Leόn, Alberto J; Xu, Luoling; Ran, Longsi; Huang, Stephen S H; Farooqui, Amber; Kelvin, David J; Kelvin, Alyson A

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus-epithelial cell interaction.

  11. The Dark Side of Cell Fusion

    PubMed Central

    Bastida-Ruiz, Daniel; Van Hoesen, Kylie; Cohen, Marie

    2016-01-01

    Cell fusion is a physiological cellular process essential for fertilization, viral entry, muscle differentiation and placental development, among others. In this review, we will highlight the different cancer cell-cell fusions and the advantages obtained by these fusions. We will specially focus on the acquisition of metastatic features by cancer cells after fusion with bone marrow-derived cells. The mechanism by which cancer cells fuse with other cells has been poorly studied thus far, but the presence in several cancer cells of syncytin, a trophoblastic fusogen, leads us to a cancer cell fusion mechanism similar to the one used by the trophoblasts. The mechanism by which cancer cells perform the cell fusion could be an interesting target for cancer therapy. PMID:27136533

  12. EP300-ZNF384 fusion gene product up-regulates GATA3 gene expression and induces hematopoietic stem cell gene expression signature in B-cell precursor acute lymphoblastic leukemia cells.

    PubMed

    Yaguchi, Akinori; Ishibashi, Takeshi; Terada, Kazuki; Ueno-Yokohata, Hitomi; Saito, Yuya; Fujimura, Junya; Shimizu, Toshiaki; Ohki, Kentaro; Manabe, Atsushi; Kiyokawa, Nobutaka

    2017-04-04

    ZNF384-related fusion genes are associated with a distinct subgroup of B-cell precursor acute lymphoblastic leukemias in childhood, with a frequency of approximately 3-4%. We previously identified a novel EP300-ZNF384 fusion gene. Patients with the ZNF384-related fusion gene exhibit a hematopoietic stem cell (HSC) gene expression signature and characteristic immunophenotype with negative or low expression of CD10 and aberrant expression of myeloid antigens, such as CD33 and CD13. However, the molecular basis of this pathogenesis remains completely unknown. In the present study, we examined the biological effects of EP300-ZNF384 expression induced by retrovirus-mediated gene transduction in an REH B-cell precursor acute lymphoblastic leukemia cell line, and observed the acquisition of the HSC gene expression signature and an up-regulation of GATA3 gene expression, as assessed by microarray analysis. In contrast, the gene expression profile induced by wild-type ZNF384 in REH cells was significantly different from that by EP300-ZNF384 expression. Together with the results of reporter assays, which revealed the enhancement of GATA3-promoter activity by EP300-ZNF384 expression, these findings suggest that EP300-ZNF384 mediates GATA3 gene expression and may be involved in the acquisition of the HSC gene expression signature and characteristic immunophenotype in B-cell precursor acute lymphoblastic leukemia cells.

  13. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    SciTech Connect

    Paquette, Stéphane G.; Banner, David; Chi, Le Thi Bao; Leon, Alberto J.; Xu, Luoling; Ran, Longsi; Huang, Stephen S.H.; Farooqui, Amber; and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  14. Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells

    PubMed Central

    Slack, J. M.; Blissard, G. W.

    2013-01-01

    Summary A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, the enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac Repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata Multicapsid Nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain. PMID:11562545

  15. Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response.

    PubMed

    Alvarez, E; Moga, E; Barquinero, J; Sierra, J; Briones, J

    2010-04-01

    Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response.

  16. Deuteron-induced fusion in various environments

    SciTech Connect

    Hale, G.M.; Talley, T.L.

    1994-04-01

    The theory of deuteron-induced fusion will be discussed, first in free space, then in muonic molecules where the Coulomb repulsion is highly screened. It will be shown how a consistent description of the d + t reactions can be obtained in these environments using R-matrix theory. We compare fusion rates obtained from the time-dependent scattering theory with those implied by the partial widths of the resonance associated with muon-catalyzed d-t fusion. Finally, some speculative comments are made about how the d + d reactions might proceed in other media, such as metallic lattices. The unusual properties of states associated with ``shadow`` poles might account for some of the strange results seen in cold fusion experiments. We emphasize that the same methods can, and should, be used to describe this situation as well as the other two well-established phenomena.

  17. Cytoplasmic calcium increase via fusion with inactivated Sendai virus induces apoptosis in human multiple myeloma cells by downregulation of c-Myc oncogene

    PubMed Central

    Jiang, Yingzhe; Saga, Kotaro; Miyamoto, Yasuhide; Kaneda, Yasufumi

    2016-01-01

    Because the emergence of drug resistance is a major limitation of current treatments for multiple myeloma (MM), it is necessary to continuously develop novel anticancer strategies. Here, using an inactivated Sendai virus (Hemagglutinating Virus of Japan; HVJ) envelope (HVJ-E), we discovered that increase of cytoplasmic Ca2+ by virus-cell fusion significantly induced apoptosis against human MM cells but not peripheral blood mononuclear cells from healthy donors. Interaction of F protein of HVJ-E with MM cells increased intracellular Ca2+ level of MMs by the induction of Ca2+ efflux from endoplasmic reticulum but not influx from extracellular region. The elevation of the Ca2+ cytoplasmic level induced SMAD1/5/8 phosphorylation and translocation into the nucleus, and SMAD1/5/8 and SMAD4 complex suppressed c-Myc transcription. Meanwhile, HVJ-E decreases S62 phosphorylation of c-Myc and promotes c-Myc protein degradation. Thus, HVJ-E-induced cell death of MM resulted from suppression of c-Myc by both destabilization of c-Myc protein and downregulation of c-Myc transcription. This study indicates that HVJ-E will be a promising tool for MM therapy. PMID:27145280

  18. Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion*

    PubMed Central

    Kondo, Naoyuki; Marin, Mariana; Kim, Jeong Hwa; Desai, Tanay M.; Melikyan, Gregory B.

    2015-01-01

    Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. PMID:25589785

  19. Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

    PubMed Central

    Zhang, Kun; Gao, Peng-Fen; Yu, Pei-Wu; Rao, Yun; Zhou, Li-Xin

    2006-01-01

    AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes’ proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes’ proliferations were remarkably increased than their parental dendritic cells. CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies. PMID:16733866

  20. Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin

    SciTech Connect

    Ishikawa, Mayuko; Ogihara, Yusuke; Miura, Masahiko

    2009-11-20

    Ionizing radiation (IR) influences cell cycle-associated events in tumor cells. We expressed the fusion protein of Azami Green (AG) and the destruction box plus nuclear localization signal of human Geminin, an inhibitor of DNA replication licensing factor, in oral tumor cells. This approach allowed us to visualize G2 arrest in living cells following irradiation. The combination of time-lapse imaging analysis allowed us to observe the nuclear envelope break down (NEBD) at early M phase, and disappearance of fluorescence (DF) at the end of M phase. The duration from NEBD to DF was not much affected in irradiated cells; however, most of daughter cells harbored double-strand breaks. Complete DF was also observed in cells exhibiting abnormal mitosis or cytokinesis. We conclude that the fluorescent Geminin probe could function as a stable cell cycle indicator irrespective of genome integrity.

  1. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  2. Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes.

    PubMed

    Lamadrid Boada, A I; Romero Aguilera, I; Terzoudi, G I; González Mesa, J E; Pantelias, G; García, O

    2013-09-18

    Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8-24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. A Molluscum contagiosum fusion protein inhibits CCL1-induced chemotaxis of cells expressing CCR8 and penetrates human neonatal foreskins: clinical applications proposed.

    PubMed

    Paslin, David A; Reykjalin, Erik; Tsadik, Elias; Schour, Lionel; Lucas, Alexander

    2015-04-01

    Inflammation in atopic dermatitis is mediated in part by the chemokine CCL1 and its receptor, CCR8. Recombinant Molluscum contagiosum viral protein (rMC148p), a cc-chemokine homolog, inhibits CCL1-induced chemotaxis of cells expressing CCR8. rMC148p was prepared using the baculovirus/Sf9 insect cell expression system. The recombinant MC148 fusion protein (rMC148fp), rMC148-TAT-6xHis, was similarly prepared by adding base sequences onto the PCR primers to fuse TAT and 6xHis to rMC148p at the carboxyl terminus. rMC148fp retains the capacity of rMC148p to inhibit CCL1-induced chemotaxis. Furthermore, unlike rMC148p, topically applied rMC148fp penetrates stratum corneum of human neonatal foreskins and concentrates along the basal and lower spinous cell layers of the epidermis. rMC148fp may be a safe and effective agent in the treatment of atopic dermatitis and other CCR8-mediated disorders.

  4. Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

    PubMed Central

    Wang, Tzu-Ming; Hilgemann, Donald W.

    2008-01-01

    We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP

  5. Characterization and spinal fusion effect of rabbit mesenchymal stem cells

    PubMed Central

    2013-01-01

    Background The surface markers of mesenchymal stem cells (MSCs) of rabbits have been reported only sporadically. However, interest in the spinal fusion effect of MSCs has risen recently. The purpose of this research was to study the surface markers and spinal fusion effect of rabbit MSCs. Results Of our rabbit MSCs, 2% expressed CD14, CD29, and CD45, 1% expressed CD90 and 97% expressed CD44. These results implied the MSCs were negative for CD14, CD29, CD45, and CD90, but positive for CD44. The surgical results showed that satisfactory fusion occurred in 10 rabbits (83%) in the study group and unsatisfactory fusion in 2 (17%). In the control group, satisfactory fusion was found in 3 rabbits (25%) and unsatisfactory fusion in 9 (75%). Statistical analysis showed the study group had significantly better spinal fusion results than the control group. Conclusions The surface markers of human and rabbit MSCs are not exactly the same. Rabbit MSCs do not have positive reactivity for CD29 and CD90, which are invariably present on human MSCs. The allogeneic undifferentiated rabbit MSCs were able to promote spinal fusion and did not induce an adverse immune response. PMID:24325928

  6. Optical imaging of cell fusion and fusion proteins in Caenorhabditis elegans.

    PubMed

    Ems, Star; Mohler, William A

    2008-01-01

    Cell fusion is a very dynamic process in which the entire membrane and cellular contents of two or more cells merge into one. Strategies developed to understand the component processes that make up a full fusion event require imaging to be performed over a range of space and time scales. These strategies must cover detection of nanometer-sized pores, monitoring cytoplasmic diffusion and the dynamic localization of proteins that induce fusion competence, and three-dimensional reconstruction of multinucleated cells. Caenorhabditis elegans' small size, predictable development, and transparent body make this organism optimal for microscopic investigations. In this chapter, focus is placed on light microscopy techniques that have been used thus far to study developmental fusion events in C. elegans and the insights that have been gained from them. There is also a general overview of the developmental timing of the cell fusion events. Additionally, several protocols are described for preparing both fixed and live specimens at various developmental stages of C. elegans for examination via optical microscopy.

  7. The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells

    PubMed Central

    Bailey, Arnold D.; Gray, Lucas T.; Pavelitz, Thomas; Newman, John C.; Horibata, Katsuyoshi; Tanaka, Kiyoji; Weiner, Alan M.

    2012-01-01

    Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43 Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome. PMID:22483866

  8. Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

    PubMed Central

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

  9. High magnetic field induced otolith fusion in the zebrafish larvae

    PubMed Central

    Pais-Roldán, Patricia; Singh, Ajeet Pratap; Schulz, Hildegard; Yu, Xin

    2016-01-01

    Magnetoreception in animals illustrates the interaction of biological systems with the geomagnetic field (geoMF). However, there are few studies that identified the impact of high magnetic field (MF) exposure from Magnetic Resonance Imaging (MRI) scanners (>100,000 times of geoMF) on specific biological targets. Here, we investigated the effects of a 14 Tesla MRI scanner on zebrafish larvae. All zebrafish larvae aligned parallel to the B0 field, i.e. the static MF, in the MRI scanner. The two otoliths (ear stones) in the otic vesicles of zebrafish larvae older than 24 hours post fertilization (hpf) fused together after the high MF exposure as short as 2 hours, yielding a single-otolith phenotype with aberrant swimming behavior. The otolith fusion was blocked in zebrafish larvae under anesthesia or embedded in agarose. Hair cells may play an important role on the MF-induced otolith fusion. This work provided direct evidence to show that high MF interacts with the otic vesicle of zebrafish larvae and causes otolith fusion in an “all-or-none” manner. The MF-induced otolith fusion may facilitate the searching for MF sensors using genetically amenable vertebrate animal models, such as zebrafish. PMID:27063288

  10. Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα

    PubMed Central

    Steinert, Gunnar; Oancea, Claudia; Roos, Jessica; Hagemeyer, Heike; Maier, Thorsten; Ruthardt, Martin; Puccetti, Elena

    2011-01-01

    Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings. PMID:21811629

  11. Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

    PubMed

    Steinert, Gunnar; Oancea, Claudia; Roos, Jessica; Hagemeyer, Heike; Maier, Thorsten; Ruthardt, Martin; Puccetti, Elena

    2011-01-01

    Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

  12. Amiodarone-induced T-U fusion.

    PubMed

    Omar, Hesham R

    2012-11-01

    Amiodarone is a widely used antiarrythmic drug for various atrial and ventricular arrhythmias. It has the potential to cause prolongation of the QT interval, which, in turn, can increases the incidence of torsade de pointes. Amiodarone is also one of the causes of prominent U waves. The presented case exemplifies the phenomenon of amiodarone-induced T-U fusion and QT prolongation. Other causes of QT prolongation as electrolyte abnormalities or administration of other drugs that prolong the QT interval were excluded. Awareness of this phenomenon and method of calculation of QT interval in this scenario is of utmost importance.

  13. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice.

    PubMed

    Wang, Yong; Yue, Xiaolin; Jin, Hongyan; Liu, Guangqing; Pan, Ling; Wang, Guijun; Guo, Hao; Li, Gang; Li, Yongdong

    2015-12-01

    Peste des petits ruminants (PPR), a highly contagious disease induced by PPR virus (PPRV), affects sheep and goats. PPRV fusion (F) protein is important for the induction of immune responses against PPRV. We constructed a Semliki Forest virus (SFV) replicon-vectored DNA vaccine ("suicidal DNA vaccine") and evaluated its immunogenicity in BALB/c mice. The F gene of PPRV was cloned and inserted into the SFV replicon-based vector pSCA1. The antigenicity of the resultant plasmid pSCA1/F was identified by indirect immunofluorescence and western blotting. BALB/c mice were then intramuscularly injected with pSCA1/F three times at 14-d intervals. Specific antibodies and virus-neutralizing antibodies against PPRV were quantified by indirect ELISA and microneutralization tests, respectively. Cell-mediated immune responses were examined by cytokine and lymphocyte proliferation assays. The pSCA1/F expressed F protein in vitro and induced specific and neutralizing antibody production, and lymphocyte proliferation in mice. Mice vaccinated with pSCA1/F had increased IL-2 and IL-10 levels after 24-h post first immunization. IFN-γ and TNF-α levels increased from that time point and gradually decreased thereafter. Thus, the Semliki Forest virus replicon-vectored DNA vaccine expressing the F protein of PPRV induced both humoral and cell-mediated immune responses in mice. This could be considered as a novel strategy for vaccine development against PPR. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Microfluidic device for high-yield pairing and fusion of stem cells with somatic cells

    NASA Astrophysics Data System (ADS)

    Gel, Murat; Hirano, Kunio; Oana, Hidehiro; Kotera, Hidetoshi; Tada, Takashi; Washizu, Masao

    2011-12-01

    Electro cell fusion has significant potential as a biotechnology tool with applications ranging from antibody production to cellular reprogramming. However due to low fusion efficiency of the conventional electro fusion methodology the true potential of the technique has not been reached. In this paper, we report a new method which takes cell fusion efficiency two orders magnitude higher than the conventional electro fusion method. The new method, based on one-toone pairing, fusion and selection of fused cells was developed using a microfabricated device. The device was composed of two microfluidic channels, a micro slit array and a petri dish integrated with electrodes. The electrodes positioned in each channel were used to generate electric field lines concentrating in the micro slits. Cells were introduced into channels and brought in to contact through the micro slit array using dielectrophoresis. The cells in contact were fused by applying a DC pulse to electrodes. As the electric field lines were concentrated at the micro slits the membrane potential was induced only at the vicinity of the micro slits, namely only at the cell-cell contact point. This mechanism assured the minimum damage to cells in the fusion as well as the ability to control the strength and location of induced membrane potential. We introduced mouse embryonic stem cells and mouse embryonic fibroblasts to the microfluidic channels and demonstrated high-yield fusion (> 80%). Post-fusion study showed the method can generate viable hybrids of stem cells and embryonic fibroblasts. Multinucleated hybrid cells adhering on the chip surface were routinely obtained by using this method and on-chip culturing.

  15. A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

    PubMed

    Motomura, Kazuki; Kawashima, Tomokazu; Berger, Frédéric; Kinoshita, Tetsu; Higashiyama, Tetsuya; Maruyama, Daisuke

    2017-08-14

    Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion by an in vitro-culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control. © 2017. Published by The Company of Biologists Ltd.

  16. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.

    PubMed

    Chudley, Lindsey; McCann, Katy; Mander, Ann; Tjelle, Torunn; Campos-Perez, Juan; Godeseth, Rosemary; Creak, Antonia; Dobbyn, James; Johnson, Bernadette; Bass, Paul; Heath, Catherine; Kerr, Paul; Mathiesen, Iacob; Dearnaley, David; Stevenson, Freda; Ottensmeier, Christian

    2012-11-01

    We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA(27-35). We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2(+) patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2(-) control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4(+) and PSMA(27)-specific CD8(+) T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4(+) T-cell response and PSMA(27)-specific CD8(+) T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4(+) and CD8(+) vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA(27) vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.

  17. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    PubMed

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. © 2014 Wiley Periodicals, Inc.

  18. Cell Fusion as a Cause of Prostate Cancer Metastases

    DTIC Science & Technology

    2011-04-01

    Fusion as a Cause of Prostate Cancer Metastases 2 14. ABSTRACT The main goal of the study funded by this grant was to test a hypothesis that cell...24 4 INTRODUCTION The main goal of this study was to test a...PC3 cultured in normal medium were used as a control (B). Some of the cells in which expression of EGFP was induced are indicated by arrows

  19. Embryonic stem cell-somatic cell fusion and postfusion enucleation.

    PubMed

    Sumer, Huseyin; Verma, Paul J

    2015-01-01

    Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.

  20. Prolonged interval between fusion and activation impairs embryonic development by inducing chromosome scattering and nuclear aneuploidy in pig somatic cell nuclear transfer.

    PubMed

    You, Jinyoung; Song, Kilyoung; Lee, Eunsong

    2010-01-01

    The aim of the present study was to examine the effect of various intervals between electrofusion and activation (FA interval) on the nuclear remodelling and development of somatic cell nuclear transfer (SCNT) embryos in pigs. Reconstructed oocytes were activated at 0 (simultaneous fusion and activation; SFA), 1, 2 and 3 h (delayed activation) after electrofusion; these groups were designated as DA1, DA2 and DA3, respectively. When oocyte nuclear status was examined at 0.5, 1, 2 and 3 h after electrofusion, the incidence of chromosome scattering was increased (P < 0.01) as the FA interval was extended (0.0%, 12.0%, 77.3% and 78.0%, respectively). Extending the FA interval led to an increase (P < 0.01) in the percentage of oocytes containing multiple (>or=3) pseudopronuclei (PPN) (0.0% of SFA; 5.3% of DA1; 21.7% of DA2; and 33.5% of DA3). The development of SCNT embryos to the blastocyst stage was decreased (P < 0.05) in DA2 (5.7%) and DA3 (5.0%) compared with SFA (18.1%) and DA1 (19.5%). Our results demonstrate that extending the FA interval impairs the development of SCNT pig embryos by inducing chromosome scattering and the formation of multiple PPN, which may result in increased nuclear aneuploidy.

  1. Genetic basis of cell-cell fusion mechanisms.

    PubMed

    Aguilar, Pablo S; Baylies, Mary K; Fleissner, Andre; Helming, Laura; Inoue, Naokazu; Podbilewicz, Benjamin; Wang, Hongmei; Wong, Melissa

    2013-07-01

    Cell-cell fusion in sexually reproducing organisms is a mechanism to merge gamete genomes and, in multicellular organisms, it is a strategy to sculpt organs, such as muscle, bone, and placenta. Moreover, this mechanism has been implicated in pathological conditions, such as infection and cancer. Studies of genetic model organisms have uncovered a unifying principle: cell fusion is a genetically programmed process. This process can be divided in three stages: competence (cell induction and differentiation); commitment (cell determination, migration, and adhesion); and cell fusion (membrane merging and cytoplasmic mixing). Recent work has led to the discovery of fusogens, which are cell fusion proteins that are necessary and sufficient to fuse cell membranes. Two unrelated families of fusogens have been discovered, one in mouse placenta and one in Caenorhabditis elegans (syncytins and F proteins, respectively). Current research aims to identify new fusogens and determine the mechanisms by which they merge membranes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Genetic basis of cell-cell fusion mechanisms

    PubMed Central

    Aguilar, Pablo S.; Baylies, Mary K.; Fleissner, Andre; Helming, Laura; Inoue, Naokazu; Podbilewicz, Benjamin; Wang, Hongmei; Wong, Melissa

    2013-01-01

    Cell-cell fusion in sexually reproducing organisms is a mechanism to merge gamete genomes, and in multicellular organisms, it is a strategy to sculpt organs such as muscles, bones, and placenta. Moreover, this mechanism has been implicated in pathological conditions such as infection and cancer. Study of genetic model organisms has uncovered a unifying principle: cell fusion is a genetically programmed process. This process can be divided in three stages: (i) competence: cell induction and differentiation, (ii) commitment: cell determination, migration and adhesion, and (iii) cell fusion: membrane merging and cytoplasmic mixing. Recent work has led to the discovery of fusogens, cell fusion proteins that are necessary and sufficient to fuse cell membranes. Two unrelated families of fusogens have been discovered, one in mouse placenta and one in Caenorhabditis elegans (Syncytins and F proteins, respectively). Current research aims to identify new fusogens and determine the mechanisms by which fusogens merge membranes. PMID:23453622

  3. Effects of ROCK inhibitor Y-27632 on cell fusion through a microslit.

    PubMed

    Wada, Ken-Ichi; Hosokawa, Kazuo; Ito, Yoshihiro; Maeda, Mizuo

    2015-11-01

    We previously reported a direct cytoplasmic transfer method using a microfluidic device, in which cell fusion was induced through a microslit (slit-through-fusion) by the Sendai virus envelope (HVJ-E) to prevent nuclear mixing. However, the method was impractical due to low efficiency of slit-through-fusion formation and insufficient prevention of nuclear mixing. The purpose of this study was to establish an efficient method for inducing slit-through-fusion without nuclear mixing. We hypothesized that modulation of cytoskeletal component can decrease nuclear migration through the microslit considering its functions. Here we report that supplementation with Y-27632, a specific ROCK inhibitor, significantly enhances cell fusion induction and prevention of nuclear mixing. Supplementation with Y-27632 increased the formation of slit-through-fusion efficiency by more than twofold. Disruption of F-actin by Y-27632 prevented nuclear migration between fused cells through the microslit. These two effects of Y-27632 led to promotion of the slit-through-fusion without nuclear mixing with a 16.5-fold higher frequency compared to our previous method (i.e., cell fusion induction by HVJ-E without supplementation with Y-27632). We also confirmed that mitochondria were successfully transferred to the fusion partner under conditions of Y-27632 supplementation. These findings demonstrate the practicality of our cell fusion system in producing direct cytoplasmic transfer between live cells. © 2015 Wiley Periodicals, Inc.

  4. Multinucleated giant cells generation induced by interferon-gamma. Changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophages fusion and multinucleated giant cell formation.

    PubMed

    Fais, S; Burgio, V L; Silvestri, M; Capobianchi, M R; Pacchiarotti, A; Pallone, F

    1994-11-01

    Multinucleated giant cells (MGC), interferon-gamma (IFN-gamma) production, and increased expression of adhesion molecules are features of granulomatous reactions. IFN-gamma induces the fusion of macrophages and the subsequent MGC generation in vitro. Moreover, IFN-gamma increases ICAM-1 expression on lymphoid cells and an important role for adhesion molecules in MGC generation has been proposed. The time course of the IFN-gamma-driven MGC generation was investigated in slide-chamber cultures of adherence-purified human monocytes. The fusion index, the monocytes clustering the total number of MGC were determined. The expression of intercellular cell adhesion molecule-1 (ICAM-1), LFA-1 and HLA-DR was investigated by immunohistochemistry. The effect of anti-ICAM-1, anti-LFA-1 and anti-HLA-DR monoclonal antibodies on IFN-gamma-induced MGC generation was also examined. IFN-gamma enhanced the generation of MGC in a dose- and time-dependent fashion. In all experiments, MGC formation was preceded by a sequence of changes in the morphology of cultured monocytes. Cell clustering occurred as early as 3 days after IFN-gamma stimulation and was followed by the adhesion of cells that eventually fused. Immunohistochemistry showed that ICAM-1 was increased by IFN-gamma and constantly polarized on a cell uropode. When monocytes clustered, ICAM-1 was localized on the membrane where the cell-to-cell contact occurred. In newly formed MGC, ICAM-1 stained in the center of the giant cell. The cellular distribution of LFA-1 on cultured monocytes was not modified by IFN-gamma. HLA-DR expectedly enhanced by IFN-gamma was mostly cytoplasmic and tended to disappear when MGC formed. Finally, anti-LFA-1 and anti-ICAM-1 monoclonal antibodies variably inhibited IFN-gamma-induced MGC generation. Taken together, these data add support to the concept that IFN-gamma is essential for MGC generation by promoting cell clustering and cell-to-cell adhesion. The present data also indicate that among the

  5. Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: Implication in multinucleation and chemosensitization

    SciTech Connect

    Park, Jeong-Eun; Woo, Seon Rang; Kang, Chang-Mo; Juhn, Kyoung-Mi; Ju, Yeun-Jin; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun Ran; Park, In-chul; Hong, Sung Hee; Hwang, Sang-Gu; Lee, Jung-Kee; Kim, Hae Kwon; Cho, Myung-Haing; Park, Gil Hong; Lee, Kee-Ho

    2011-01-14

    Research highlights: {yields} Paclitaxel serves as a stimulator of chromosomal fusion in cells in which telomeres are dysfunctional. {yields} Typical fusions involve p-arms, but paclitaxel-induced fusions occur between both q- and p-arms. {yields} Paclitaxel-stimulated fusions in cells in which telomeres are dysfunctional evoke prolonged G2/M cell cycle arrest and delay multinucleation. {yields} Upon telomere erosion, paclitaxel promotes chromosomal instability and subsequent apoptosis. {yields} Chromosomal fusion enhances paclitaxel chemosensitivity under telomere dysfunction. -- Abstract: The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC-/-p53-/- mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.

  6. Khz (fusion product of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in human colon carcinoma HCT116 cells, accompanied by an increase in reactive oxygen species, activation of caspase 3, and increased intracellular Ca²⁺.

    PubMed

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2015-03-01

    Khz (a fusion mycelium of Ganoderma lucidum and Polyporus umbellatus mycelia) is isolated from ganoderic acid and P. umbellatus and it exerts antiproliferative effects against malignant cells. However, no previous study has reported the inhibitory effects of Khz on the growth of human colon cancer cells. In the present study, we found that Khz suppressed cell division and induced apoptosis in HCT116 cells. Khz cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Khz reduced cell viability and mitochondrial membrane potential levels and it also induced disruption of the mitochondrial membrane potential and increased calcium concentration and reactive oxygen species generation. Khz increased caspase 3, PARP, caspase 7, and caspase 9 levels, but reduced Bcl-2 protein levels. Flow cytometry showed that the percentage of HCT116 cells in the sub-G1 phase of the cell cycle increased in response to Khz treatment.

  7. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    NASA Astrophysics Data System (ADS)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  8. Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

    PubMed Central

    Horvath, C M; Lamb, R A

    1992-01-01

    The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed. Images PMID:1548771

  9. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2007-03-01

    Development 109:833–844, 1990. 28Marx, R. E.. Clinical application of bone biology to man- dibular and maxillary reconstruction. Clin. Plast. Surg. 21...in sympathetic neuron development . Drug News Perspect 16, 589, 2003. 8. Cayuso, J., and Marti, E. Morphogens in motion: growth control of the...this study is to develop a safe efficacious system for inducing spine fusion which will eliminate the need for invasive surgery. We have currently

  10. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

    PubMed Central

    Zheng, Yi-Min; Melikyan, Gregory B.; Liu, Shan-Lu; Cohen, Fredric S.

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  11. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    PubMed

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

  12. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    SciTech Connect

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang; Liu, Yan-Hong; Li, Yan; Wang, Jia-Ye; Hattori, Toshio; Ling, Hong; Zhang, Feng-Min

    2010-01-22

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  13. Synaptotagmin IV Modulation of Vesicle Size and Fusion Pores in PC12 Cells

    PubMed Central

    Zhang, Zhenjie; Zhang, Zhen; Jackson, Meyer B.

    2010-01-01

    Many synaptotagmins are Ca2+-binding membrane proteins with functions in Ca2+-triggered exocytosis. Synaptotagmin IV (syt IV) has no Ca2+ binding activity, but nevertheless modulates exocytosis. Here, cell-attached capacitance recording was used to study single vesicle fusion and fission in control and syt IV overexpressing PC12 cells. Unitary capacitance steps varied widely in size, indicating that both microvesicles (MVs) and dense-core vesicles (DCVs) undergo fusion. Syt IV overexpression reduced the size of DCVs and endocytotic vesicles but not MVs. Syt IV also reduced the basal rate of Ca2+-induced fusion. During kiss-and-run, syt IV increased the conductance and duration of DCV fusion pores but not MV fusion pores. During full-fusion of DCVs syt IV increased the fusion pore conductance but not the duration. Syt IV overexpression increased the duration but not the conductance of fission pores during endocytosis. The effects of syt IV on fusion pores in PC12 cells resembled the effects on fusion pores in peptidergic nerve terminals. However, differences between these and results obtained with amperometry may indicate that amperometry and capacitance detect the fusion of different populations of vesicles. The effects of syt IV on fusion pores are discussed in terms of structural models and kinetic mechanisms. PMID:20303854

  14. Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.

    PubMed

    Tsurudome, M; Ito, Y

    2000-01-01

    Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.

  15. What Makes Fusion Cells Effective?

    DTIC Science & Technology

    2009-12-01

    cells, one of the key works is Henry Mintzberg’s 1980 article “ Organizational Design: Fashion or Fit?” In this article, Mintzberg discusses the...characterized by mutual adjustment have better outcomes. The leadership style found in FCs will strongly influence the organizational design structure and... ORGANIZATIONAL DESIGN LITERATURE........................................11 E. LITERATURE FROM U.S. GOVERNMENT OR PUBLIC ORGANIZATIONS FOCUSED ON

  16. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

    PubMed Central

    Kim, Irene S.; Jenni, Simon; Stanifer, Megan L.; Roth, Eatai; Whelan, Sean P. J.; van Oijen, Antoine M.; Harrison, Stephen C.

    2017-01-01

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion. PMID:27974607

  17. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein.

    PubMed

    Kim, Irene S; Jenni, Simon; Stanifer, Megan L; Roth, Eatai; Whelan, Sean P J; van Oijen, Antoine M; Harrison, Stephen C

    2017-01-03

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a "class I" fusogen) and West Nile virus envelope protein ("class II"). Our study of VSV now extends this description to "class III" viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

  18. Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation.

    PubMed

    Mateo-Lozano, Silvia; Tirado, Oscar M; Notario, Vicente

    2003-12-18

    Ewing's sarcoma (ES) is the prototype of a family of tumors (ESFT) of neuroectodermal origin formed by small, round cells with limited neural differentiation, which arise most frequently within bones in children or adolescents. The proliferation of ESFT cells is highly dependent on the establishment of, and signaling through several growth factor-mediated autocrine loops. The mammalian target of rapamycin (mTOR) is a central regulator of translation and cell proliferation, involved in the cellular response to various nutritional, stress and mitogenic effectors. As mTOR has recently been associated with certain human cancers, we investigated the possibility that mTOR played a role in the regulation of ES cell proliferation. Results showed that ES cell lines carrying EWS/FLI-1 alleles of different types expressed different levels of total and phosphorylated mTOR protein. We demonstrate that rapamycin, an mTOR inhibitor, efficiently blocked the proliferation of all cell lines by promoting cell cycle arrest at the G1 phase. This was paralleled by the downregulation of the levels of the EWS/FLI-1 proteins, regardless of their fusion type, and the concomitant restoration of the expression of the TGF-beta type 2 receptor (TGFbeta RII), which is known to be repressed by several EWS-ETS fusion proteins. The expression of a rapamycin-resistant mTOR construct prevented both the proliferation blockade and the EWS/FLI-1 downregulation. These data demonstrate that mTOR signaling plays a central role in ES cell pathobiology and strongly suggest that the use of rapamycin as a cytostatic agent may be an efficient tool for the treatment of ES patients.

  19. Resveratrol Regulates Mitochondrial Biogenesis and Fission/Fusion to Attenuate Rotenone-Induced Neurotoxicity

    PubMed Central

    Peng, Kaige; Tao, Yuan; Zhang, Jun; Wang, Jian; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Cai, Ying; Zhao, Jiqing; Wu, Qiang; Zou, Zhongmin; Cao, Jia; Sai, Yan

    2016-01-01

    It has been confirmed that mitochondrial impairment may underlie both sporadic and familial Parkinson's disease (PD). Mitochondrial fission/fusion and biogenesis are key processes in regulating mitochondrial homeostasis. Therefore, we explored whether the protective effect of resveratrol in rotenone-induced neurotoxicity was associated with mitochondrial fission/fusion and biogenesis. The results showed that resveratrol could not only promote mitochondrial mass and DNA copy number but also improve mitochondrial homeostasis and neuron function in rats and PC12 cells damaged by rotenone. We also observed effects with alterations in proteins known to regulate mitochondrial fission/fusion and biogenesis in rotenone-induced neurotoxicity. Therefore, our findings suggest that resveratrol may prevent rotenone-induced neurotoxicity through regulating mitochondrial fission/fusion and biogenesis. PMID:26770656

  20. Osteoclasts and giant cells: macrophage–macrophage fusion mechanism

    PubMed Central

    Vignery, Agnès

    2000-01-01

    Membrane fusion is a ubiquitous event that occurs in a wide range of biological processes. While intracellular membrane fusion mediating organelle trafficking is well understood, much less is known about cell–cell fusion mediating sperm cell–oocyte, myoblast–myoblast and macrophage–macrophage fusion. In the case of mononuclear phagocytes, their fusion is not only associated with the differentiation of osteoclasts, cells which play a key role in the pathogenesis of osteoporosis, but also of giant cells that are present in chronic inflammatory reactions and in tumours. Despite the biological and pathophysiological importance of intercellular fusion events, the actual molecular mechanism of macrophage fusion is still unclear. One of the main research themes in my laboratory has been to investigate the molecular mechanism of mononuclear phagocyte fusion. Our hypothesis has been that macrophage–macrophage fusion, similar to virus–cell fusion, is mediated by specific cell surface proteins. But, in contrast with myoblasts and sperm cells, macrophage fusion is a rare event that occurs in specific instances. To test our hypothesis, we established an in vitro cell–cell fusion assay as a model system which uses alveolar macrophages. Upon multinucleation, these macrophages acquire the osteoclast phenotype. This indicates that multinucleation of macrophages leads to a specific and novel functional phenotype in macrophages. To identify the components of the fusion machinery, we generated four monoclonal antibodies (mAbs) which block the fusion of alveolar macrophages and purified the unique antigen recognized by these mAbs. This led us to the cloning of MFR (Macrophage Fusion Receptor). MFR was cloned simultaneously as P84/SHPS-1/SIRPα/BIT by other laboratories. We subsequently showed that the recombinant extracellular domain of MFR blocks fusion. Most recently, we identified a lower molecular weight form of MFR that is missing two extracellular immunoglobulin (Ig

  1. Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

    PubMed Central

    Hardy, Gregory J.; Nayak, Rahul

    2013-01-01

    Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle-vesicle and vesicle-substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates. PMID:24031164

  2. Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed-Sternberg cells.

    PubMed

    Rengstl, Benjamin; Newrzela, Sebastian; Heinrich, Tim; Weiser, Christian; Thalheimer, Frederic B; Schmid, Frederike; Warner, Kathrin; Hartmann, Sylvia; Schroeder, Timm; Küppers, Ralf; Rieger, Michael A; Hansmann, Martin-Leo

    2013-12-17

    Multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well.

  3. Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed–Sternberg cells

    PubMed Central

    Rengstl, Benjamin; Newrzela, Sebastian; Heinrich, Tim; Weiser, Christian; Thalheimer, Frederic B.; Schmid, Frederike; Warner, Kathrin; Hartmann, Sylvia; Schroeder, Timm; Küppers, Ralf; Rieger, Michael A.; Hansmann, Martin-Leo

    2013-01-01

    Multinucleated Reed–Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well. PMID:24302766

  4. Inflammation and Proliferation Act Together to Mediate Intestinal Cell Fusion

    PubMed Central

    Swain, John R.; Wong, Melissa H.

    2009-01-01

    Cell fusion between circulating bone marrow-derived cells (BMDCs) and non-hematopoietic cells is well documented in various tissues and has recently been suggested to occur in response to injury. Here we illustrate that inflammation within the intestine enhanced the level of BMDC fusion with intestinal progenitors. To identify important microenvironmental factors mediating intestinal epithelial cell fusion, we performed bone marrow transplantation into mouse models of inflammation and stimulated epithelial proliferation. Interestingly, in a non-injury model or in instances where inflammation was suppressed, an appreciable baseline level of fusion persisted. This suggests that additional mediators of cell fusion exist. A rigorous temporal analysis of early post-transplantation cellular dynamics revealed that GFP-expressing donor cells first trafficked to the intestine coincident with a striking increase in epithelial proliferation, advocating for a required fusogenic state of the host partner. Directly supporting this hypothesis, induction of augmented epithelial proliferation resulted in a significant increase in intestinal cell fusion. Here we report that intestinal inflammation and epithelial proliferation act together to promote cell fusion. While the physiologic impact of cell fusion is not yet known, the increased incidence in an inflammatory and proliferative microenvironment suggests a potential role for cell fusion in mediating the progression of intestinal inflammatory diseases and cancer. PMID:19657387

  5. Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

    PubMed

    Maruyama, Daisuke; Völz, Ronny; Takeuchi, Hidenori; Mori, Toshiyuki; Igawa, Tomoko; Kurihara, Daisuke; Kawashima, Tomokazu; Ueda, Minako; Ito, Masaki; Umeda, Masaaki; Nishikawa, Shuh-Ichi; Groß-Hardt, Rita; Higashiyama, Tetsuya

    2015-05-07

    In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

  6. Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells

    PubMed Central

    He, Ke; Qu, Hu; Xu, Li-Nan; Gao, Jun; Cheng, Fu-Yi; Xiang, Peng; Zhou, Can-Quan

    2016-01-01

    To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer. PMID:27377320

  7. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    SciTech Connect

    Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin; Liu, Ke; Shang, Zheng-jun

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  8. Phenylarsine oxide-induced increase in alveolar macrophage surface receptors: evidence for fusion of internal receptor pools with the cell surface

    PubMed Central

    1985-01-01

    Rabbit alveolar macrophages which were treated at 0 degrees C with phenylarsine oxide and then incubated at 37 degrees C for 10 min exhibited a two- to threefold increase in surface receptor activity for macroglobulin.protease complexes, diferric transferrin, and mannose- terminal glycoproteins. Analysis of the concentration-dependence of ligand binding indicated that changes in ligand-binding activity were due to changes in receptor number rather than alterations in ligand- receptor affinity. Surface receptor number could also be increased by treatment of cells with three other sulfhydryl reagents, N- ethylmaleimide, p-chloromercurobenzoate, and iodoacetic acid. The increase in receptor activity was maximal after 10 min and decreased over the next hour. This decrease in cell-associated receptor activity was due to the release of large membrane vesicles which demonstrated a uniform buoyant density by isopycnic sucrose gradient centrifugation. Treatment of cells with phenylarsine oxide did not decrease the cellular content of lactate dehydrogenase or beta-galactosidase, indicating that cell integrity was maintained and lysosomal enzyme release did not occur. Our studies indicate that phenylarsine oxide treatment in the presence of extracellular Ca2+ results in the fusion of receptor-containing vesicles with the cell surface. PMID:2409094

  9. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

    PubMed Central

    Ivanovic, Tijana; Choi, Jason L; Whelan, Sean P; van Oijen, Antoine M; Harrison, Stephen C

    2013-01-01

    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI: http://dx.doi.org/10.7554/eLife.00333.001 PMID:23550179

  10. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

    PubMed Central

    Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

    2010-01-01

    Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

  11. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

    SciTech Connect

    Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Ellis Dutch, Rebecca; McCann, Richard O.

    2010-08-15

    Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.

  12. Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus.

    PubMed

    Wiener, Dominique; Plattet, Philippe; Cherpillod, Pascal; Zipperle, Ljerka; Doherr, Marcus G; Vandevelde, Marc; Zurbriggen, Andreas

    2007-11-01

    Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

  13. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection

    PubMed Central

    Singh, Amit K.; Kingston, Joseph J.; Gupta, Shishir K.; Batra, Harsh V.

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100–270 aa) and YopE (50–213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

  14. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection.

    PubMed

    Singh, Amit K; Kingston, Joseph J; Gupta, Shishir K; Batra, Harsh V

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens.

  15. Kinetic Modeling of Laser-Induced Fusion

    DTIC Science & Technology

    2007-09-01

    Thermal neutrons are of considerable interest to the Department of Defense and for commercial applications. Unlike high- energy photons, neutrons easily...develop a compact generator for thermal neutrons with large enough flux. The limited availability of radio-isotopes, combined with the relatively...Deuterium-Tritium (D-T) fusion, which generates Alpha particles and fast neutrons . In these sources, Deuterium ions are accelerated to about 130 keV and hit

  16. High-Frequency Gravitational Wave Induced Nuclear Fusion

    SciTech Connect

    Fontana, Giorgio; Baker, Robert M. L. Jr.

    2007-01-30

    Nuclear fusion is a process in which nuclei, having a total initial mass, combine to produce a single nucleus, having a final mass less than the total initial mass. Below a given atomic number the process is exothermic; that is, since the final mass is less than the combined initial mass and the mass deficit is converted into energy by the nuclear fusion. On Earth nuclear fusion does not happen spontaneously because electrostatic barriers prevent the phenomenon. To induce controlled, industrial scale, nuclear fusion, only a few methods have been discovered that look promising, but net positive energy production is not yet possible because of low overall efficiency of the systems. In this paper we propose that an intense burst of High Frequency Gravitational Waves (HFGWs) could be focused or beamed to a target mass composed of appropriate fuel or target material to efficiently rearrange the atomic or nuclear structure of the target material with consequent nuclear fusion. Provided that efficient generation of HFGW can be technically achieved, the proposed fusion reactor could become a viable solution for the energy needs of mankind and alternatively a process for beaming energy to produce a source of fusion energy remotely - even inside solid materials.

  17. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.

  18. Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus.

    PubMed Central

    Obeid, O E; Partidos, C D; Howard, C R; Steward, M W

    1995-01-01

    Synthetic peptides representing T- and B-cell epitopes from the fusion (F) protein of measles virus (MV) were tested for their ability to induce a protective immune response against intracerebral challenge with neuroadapted strains of MV and canine distemper virus (CDV) in mice. Of the panel of peptides tested, only a chimeric peptide consisting of two copies of a promiscuous T-cell epitope (representing residues 288 to 302 of MV F protein) synthesized at the amino terminus of a B-cell epitope (representing residues 404 to 414 of MV F protein) was able to induce a protective response against challenge with MV and CDV in inbred mice. The protective response induced by this peptide (TTB) was associated with a significant reduction in mortality, histological absence of acute encephalitis, and greatly reduced titers of virus in the brains of TTB-immune mice following challenge compared with the results for nonimmunized controls. A chimeric peptide comprising one copy of the T-cell epitope and one copy of the B-cell epitope (TB) did not induce a protective response. A comparison of the antibody responses induced by the two chimeras suggested that differences in protective efficacy following immunization may be a result of the higher affinity of the antibody induced by the TTB peptide than that of the antibody induced by the TB peptide. In addition, differences in the immunoglobulin G subclass of the antipeptide antibody responses were observed, and these may play a role in the differences in protection observed. These results indicate that appropriately designed synthetic peptides have potential as vaccines for the induction of cross-reactive protection against morbilliviruses. PMID:7531779

  19. Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell-cell fusion assays and in response to membrane curvature agents

    SciTech Connect

    Clancy, Eileen K.; Barry, Chris; Ciechonska, Marta; Duncan, Roy

    2010-02-05

    The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.

  20. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

  1. Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study.

    PubMed

    Lewis, Kenneth T; Naik, Akshata R; Laha, Suvra S; Wang, Sunxi; Mao, Guangzhao; Kuhn, Eric; Jena, Bhanu P

    2017-08-03

    A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Fusion of Ubiquitin to HIV Gag Impairs Human Monocyte-Derived Dendritic Cell Maturation and Reduces Ability to Induce Gag T Cell Responses

    PubMed Central

    Herath, Shanthi; Benlahrech, Adel; Papagatsias, Timos; Athanasopoulos, Takis; Bouzeboudjen, Zineb; Hervouet, Catherine; Klavinskis, Linda; Meiser, Andrea; Kelleher, Peter; Dickson, George; Patterson, Steven

    2014-01-01

    The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs. PMID:24505475

  3. Mitochondrial fusion and division: Regulation and role in cell viability.

    PubMed

    Benard, Giovanni; Karbowski, Mariusz

    2009-05-01

    Discovery of various molecular components regulating dynamics and organization of the mitochondria in cells, together with novel insights into the role of mitochondrial fusion and division in the maintenance of cellular homeostasis, have provided some of the most exciting breakthroughs in the last decade of mitochondrial research. The focus of this review is on the regulation of mitochondrial fusion and division machineries. The newly identified factors associated with mitofusin/OPA1-dependent mitochondrial fusion, and Drp1-dependent mitochondrial division are discussed. Furthermore, the most recent findings on the role of mitochondrial fusion and division in the maintenance of cell function are also reviewed here in some detail.

  4. Oral administration of a non-absorbable plant cell-expressed recombinant anti-TNF fusion protein induces immunomodulatory effects and alleviates nonalcoholic steatohepatitis

    PubMed Central

    Ilan, Yaron; Ben Ya'acov, Ami; Shabbat, Yehudit; Gingis-Velitski, Svetlana; Almon, Einat; Shaaltiel, Yoseph

    2016-01-01

    AIM To evaluate the immunomodulatory effect of oral administration of PRX-106 in the high-fat diet model. METHODS For 22 wk, C57BL/6 HFD-fed mice received daily oral treatments with BY-2 cells expressing recombinant anti-tumor necrosis factor alpha fusion protein (PRX-106). Mice were followed for serum liver enzyme and triglyceride levels, liver histology and intrahepatic and systemic FACS. RESULTS The orally administered non-absorbable PRX-106 was biologically active. Altered distribution of CD4+CD25+FoxP3+ between the liver and spleen and an increase in the intrasplenic-to-intrahepatic CD4+CD25+FoxP3+ ratio and a decrease in the intrasplenic-to-intrahepatic CD8+CD25+FoxP3+ ratio were observed. An increase in intrahepatic NKT cells and a decrease in the intrasplenic-to-intrahepatic NKT ratio were noted. Assessment of the CD4-to-CD8 ratios showed sequestration of CD8+ lymphocytes in the liver. These effects were associated with a decrease in serum triglyceride levels, decrease in the aspartate aminotransferase levels, serum glucose levels, and HOMA-IR score. A decrease in hepatic triglycerides content was observed in the high dose-treated mice. CONCLUSION Orally administered PRX-106 shows biological activity and exerts an immunomodulatory effect, alleviating liver damage. The data suggest that PRX-106 may provide an oral immunotherapy for nonalcoholic steatohepatitis. PMID:27818591

  5. Cell fusion: biological perspectives and potential for regenerative medicine.

    PubMed

    Alvarez-Dolado, Manuel

    2007-01-01

    Cell fusion has emerged as a powerful subject of debate in the last few years. Adult stem cell plasticity and the search for mechanisms to explain this process have led to the "rediscovery" of cell fusion. In nature, cell fusion is a normal process involved in sexual reproduction, tissue formation, and immune response. The recent observation that bone marrow derived cells fuse with several cell types introduces new and provocative questions. In this review, I shall recapitulate what is known about cell fusion and discuss its more controversial aspects. I shall highlight the most exciting open questions; its biological potential; pros and cons; and their implications on stem cell plasticity, regenerative medicine, and development.

  6. Entry of enveloped viruses into host cells: membrane fusion.

    PubMed

    Más, Vicente; Melero, José A

    2013-01-01

    Viruses are intracellular parasites that hijack the cellular machinery for their own replication. Therefore, an obligatory step in the virus life cycle is the delivery of the viral genome inside the cell. Enveloped viruses (i.e., viruses with a lipid envelope) use a two-step procedure to release their genetic material into the cell: (i) they first bind to specific surface receptors of the target cell membrane and then, (ii) they fuse the viral and cell membranes. This last step may occur at the cell surface or after internalization of the virus particle by endocytosis or by some other route (e.g., macropinocytosis). Remarkably, the virus-cell membrane fusion process goes essentially along the same intermediate steps as other membrane fusions that occur for instance in vesicular fusion at the nerve synapsis or cell-cell fusion in yeast mating. Specialized viral proteins, fusogens, promote virus-cell membrane fusion. The viral fusogens experience drastic structural rearrangements during fusion, liberating the energy required to overcome the repulsive forces that prevent spontaneous fusion of the two membranes. This chapter describes the different types of viral fusogens and their mode of action, as are currently known.

  7. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  8. Event induced bias in label fusion

    NASA Astrophysics Data System (ADS)

    Schubert Kabban, Christine M.; Venzin, Alexander M.; Oxley, Mark E.

    2017-05-01

    In a two class label scenario, classification systems may be used to assess whether or not an element of interest belongs to the `target'or `non-target'class. The performance of the system is summarized visually as a trade- o¤ between the proportions of elements correctly labeled as `target'plotted against the proportion of elements incorrectly labeled as `target.' These proportions are empirical estimates of the true and false positive rates, and their trade-o¤ plot is known as a receiver operating characteristic (ROC) curve. Classification performance can be increased, however, if the information provided by multiple systems can be fused together to create a new, combined system. This research focuses on label-fusion as a common method to increase classification performance and quantifying the bias that occurs when misspecifying the partitioning of the underlying event set. This partitioning will be defined in terms of what be called within and across label fusion. When incorrect assumptions are made about the partitioning of the event set, bias will occur and both the ROC curve and its optimal parameters will be incorrectly quantified. In this work, we analyze the e¤ects of individual classification system performance, correlation, and target environment on the magnitude of this performance bias. This work will then inspire the development of formulas to adjust optimal performance measures to appropriately reflect the fused system performance according to event set partitioning. As such, bias may be appropriately adjusted without redesigning the fused system, allowing greater use of currently fused systems across multiple platforms and environments.

  9. Dendritic-tumor fusion cells in cancer immunotherapy.

    PubMed

    Takakura, Kazuki; Kajihara, Mikio; Ito, Zensho; Ohkusa, Toshifumi; Gong, Jianlin; Koido, Shigeo

    2015-03-01

    A promising area of clinical investigation is the use of cancer immunotherapy to treat cancer patients. Dendritic cells (DCs) operate as professional antigen-presenting cells (APCs) and play a critical role in the induction of antitumor immune responses. Thus, DC-based cancer immunotherapy represents a powerful strategy. One DC-based cancer immunotherapy strategy that has been investigated is the administration of fusion cells generated with DCs and whole tumor cells (DC-tumor fusion cells). The DC-tumor fusion cells can process a broad array of tumor-associated antigens (TAAs), including unidentified molecules, and present them through major histocompatibility complex (MHC) class I and II pathways in the context of co-stimulatory signals. Improving the therapeutic efficacy of DC-tumor fusion cell-based cancer immunotherapy requires increased immunogenicity of DCs and whole tumor cells. We discuss the potential ability of DC-tumor fusion cells to activate antigen-specific T cells and strategies to improve the immunogenicity of DC-tumor fusion cells as anticancer vaccines.

  10. Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

    PubMed Central

    Holm, Christian K; Jensen, Søren B; Jakobsen, Martin R; Cheshenko, Natalia; Horan, Kristy A; Moeller, Hanne B; Gonzalez-Dosal, Regina; Rasmussen, Simon B; Christensen, Maria H.; Yarovinsky, Timur O; Rixon, Frazer J; Herold, Betsy C; Fitzgerald, Katherine A; Paludan, Søren R

    2012-01-01

    The innate immune system senses infection by detecting evolutionarily conserved molecules essential for microbial survival or abnormal location of molecules. Here we demonstrate the existence of a novel innate detection mechanism, which is induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon (IFN) response with expression of IFN-stimulated genes (ISGs), in vivo recruitment of leukocytes, and potentiation of Toll-like receptor 7 and 9 signaling. The fusion dependent response was dependent on stimulator of interferon genes (STING) but independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant cell formation. PMID:22706339

  11. Effects of PIP2 on membrane fusion between mast cell SNARE liposomes mediated by synaptotagmin 2.

    PubMed

    Tadokoro, Satoshi; Inoh, Yoshikazu; Nakanishi, Mamoru; Hirashima, Naohide

    2015-10-01

    Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca2+ sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca2+ and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca2+, synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca2+, when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

    PubMed

    Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-Lin; Liu, Ke; Shang, Zheng-Jun

    2014-10-15

    Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells

    EPA Science Inventory

    Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in...

  14. Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells

    EPA Science Inventory

    Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in...

  15. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  16. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies.

    PubMed

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC(50) of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors.

  17. In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties

    PubMed Central

    1992-01-01

    We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA

  18. Cell Fusion in the War on Cancer: A Perspective on the Inception of Malignancy

    PubMed Central

    Platt, Jeffrey L.; Zhou, Xiaofeng; Lefferts, Adam R.; Cascalho, Marilia

    2016-01-01

    Cell fusion occurs in development and in physiology and rarely in those settings is it associated with malignancy. However, deliberate fusion of cells and possibly untoward fusion of cells not suitably poised can eventuate in aneuploidy, DNA damage and malignant transformation. How often cell fusion may initiate malignancy is unknown. However, cell fusion could explain the high frequency of cancers in tissues with low underlying rates of cell proliferation and mutation. On the other hand, cell fusion might also engage innate and adaptive immune surveillance, thus helping to eliminate or retard malignancies. Here we consider whether and how cell fusion might weigh on the overall burden of cancer in modern societies. PMID:27420051

  19. pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

    PubMed

    Desai, Tanay M; Marin, Mariana; Mason, Caleb; Melikyan, Gregory B

    2017-05-12

    Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

    PubMed Central

    Grassi, Francesca; Pagani, Francesca; Spinelli, Gabriele; Angelis, Luciana De; Cossu, Giulio; Eusebi, Fabrizio

    2004-01-01

    Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment. PMID:15319417

  1. Proteolytic Cleavage of the Fusion Protein but Not Membrane Fusion Is Required for Measles Virus-Induced Immunosuppression In Vitro

    PubMed Central

    Weidmann, Armin; Maisner, Andrea; Garten, Wolfgang; Seufert, Marion; ter Meulen, Volker; Schneider-Schaulies, Sibylle

    2000-01-01

    Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F0 protein by cellular proteases, such as furin, into the F1-F2 subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F0 proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0 proteins was not due to an impaired F0-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F0 protein. PMID:10644371

  2. Fusion of human bone hemopoietic stem cell with esophageal carcinoma cells didn't generate esophageal cancer stem cell.

    PubMed

    Fan, H; Lu, S

    2014-01-01

    Prior studies showed that cell fusion between bone marrow-derived cell (BMDC) and somatic cell might be the origin of cancer stem cell. Our previous study suggested that cell fusion of human bone marrow-derived mesenchymal stem cell (MSC) with esophageal cancer cell did not generate cancer stem cells. But up to now, the origin of cancer stem cell is still ambiguous. In this study, we carried out the cell fusion experiment between hemopoietic stem cells (HSCs) and human esophageal cancer cells, and found that cell fusion slowed the growth speed of esophageal cancer cells and decreased the clone formation ability and tumorigenicity in NOD/SCID mice. In addition, cell fusion did not increase the ratio of side population (SP) cells and the resistance to chemotherapeutic drugs. Collectively, our data indicated that cell fusion between HSCs and esophageal cancer cells has a therapeutic effect rather than generate cells with characteristics of esophageal cancer stem cells.

  3. β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype

    PubMed Central

    Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R.

    2016-01-01

    Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow−derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes. Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment. With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP. After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes. PMID:27374092

  4. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.

    PubMed

    Corcoran, Jennifer A; Duncan, Roy

    2004-04-01

    Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.

  5. Electrochemically induced deuterium-tritium fusion power reactor; Preliminary design of a reactor system

    SciTech Connect

    Oka, Y.; Koshizuka, S.; Kondo, S. . Faculty of Engineering )

    1989-09-01

    Conceptual design of an electrochemically induced deuterium-tritium fusion power reactor has been carried out. A double-tube-type fuel cell is proposed for efficient electrolysis and to provide a large cathode area. The fuel cell tubes are assembled like a pressurized water reactor (PWR) control rod cluster. The tritium fuel is continuously fed through the cluster rod to the cell. The voltage for the electrolysis is supplied through the rod. The tritium breeding Li/sub 2/O is contained in a hexagonal blanket through which coolant tubes penetrate. The fuel cell tube is inserted in the coolant tube and the water coolant flows through the annuli.

  6. Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

    PubMed

    Huerta, Leonor; Lamoyi, Edmundo; Báez-Saldaña, Armida; Larralde, Carlos

    2002-02-01

    In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion. Copyright 2002 Wiley-Liss, Inc.

  7. Elcatonin prevents bone loss caused by skeletal unloading by inhibiting preosteoclast fusion through the unloading-induced high expression of calcitonin receptors in bone marrow cells.

    PubMed

    Tsukamoto, Manabu; Menuki, Kunitaka; Murai, Teppei; Hatakeyama, Akihisa; Takada, Shinichiro; Furukawa, Kayoko; Sakai, Akinori

    2016-04-01

    This study aimed to clarify whether elcatonin (EL) has a preventive action on bone dynamics in skeletal unloading. Seven-week-old male C57BL/6J mice with either ground control (GC) or tail suspension (TS) were administered EL 20U/kg or a vehicle (veh) three times per week and assigned to one of the following four groups: GCEL, GCveh, TSEL, and TSveh. Blood samples and bilateral femurs and tibias of the mice were obtained for analysis. After 7days of unloading, the trabecular bone mineral density in the distal femur obtained via peripheral quantitative computed tomography and the trabecular bone volume were significantly higher in the TSEL group than in the TSveh group. The bone resorption histomorphometric parameters, such as the osteoclast surface and osteoclast number, were significantly suppressed in the TSEL mice, whereas the number of preosteoclasts was significantly increased. The plasma level of tartrate-resistant acid phosphatase-5b (TRACP-5b) was significantly lower in the TSEL group than in all other groups. In the bone marrow cell culture, the number of TRACP-positive (TRACP(+)) multinucleated cells was significantly lower in the TSEL mice than in the TSveh mice, whereas the number of TRACP(+) mononucleated cells was higher in the TSEL mice. On day 4, the expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), cathepsin K and d2 isoform of vacuolar ATPase V0 domain (ATP6V0D2) mRNA in the bone marrow cells in the TSEL mice was suppressed, and the expression of calcitonin receptor (Calcr) mRNA on day 1 and Calcr antigen on day 4 were significantly higher in the TSveh mice than in the GCveh mice. EL prevented the unloading-induced bone loss associated with the high expression of Calcr in the bone marrow cells of mouse hindlimbs after tail suspension, and it suppressed osteoclast development from preosteoclasts to mature osteoclasts through bone-resorbing activity. This study of EL-treated unloaded mice provides the

  8. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    EPA Science Inventory

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  9. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    EPA Science Inventory

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  10. Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

    PubMed Central

    Huang, S K; Cheng, M; Hui, S W

    1990-01-01

    Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values. Images FIGURE 3 FIGURE 5 FIGURE 7 PMID:2291938

  11. Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

    PubMed Central

    2014-01-01

    Background Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. Results We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. Conclusions The rCTB protein fused to a functional peptide secreted by L. casei

  12. Human HSP70 and modified HPV16 E7 fusion DNA vaccine induces enhanced specific CD8+ T cell responses and anti-tumor effects.

    PubMed

    Zong, Jinbao; Peng, Qinglin; Wang, Qingyong; Zhang, Ting; Fan, Dongsheng; Xu, Xuemei

    2009-10-01

    Cervical cancer is one of the most common cancers in women worldwide and persistent infection with human papilloma virus (HPV)s is considered to be the major risk factor. Millions of women are currently infected with high risk genotypes. Therefore, it is imperative to develop therapeutic vaccines to eliminate established infection or HPV-related disease. In the current study, we generated two potential therapeutic HPV DNA vaccines, SigmE7/MtHSP70 and SigmE7/HuHSP70, using human and mycobacterium tuberculosis HSP70 linked, respectively, to HPV16 mE7 and the signal peptide gene of human CD33. Our comparative evaluation of these two vaccines found that vaccination with the SigmE7/HuHSP70 DNA vaccine induced a stronger E7-specific CD8+ T cell immune response and resulted in a more significant therapeutic effect against E7 expressing tumor cells. Our study may serve as an important foundation and significant reference for future clinical applications.

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

    PubMed

    Dudin, Omaya; Merlini, Laura; Martin, Sophie G

    2016-10-01

    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.

  14. Optical manipulation of hot nanoparticles can mediate selected cell fusion

    NASA Astrophysics Data System (ADS)

    Oddershede, Lene B.; Bahadori, Azra; Bendix, Poul M.

    2017-04-01

    Metallic nanoparticles with diameters from 10 nm to 250 nm can be optically trapped and manipulated in 3D using a single tightly focused near infrared laser beam. This will result in a significant heating of the particle and its vicinity, with temperature increases easily reaching hundreds degrees Celsius. If such a hot metallic nanoparticle is brought into the contact zone between two cells or vesicles, this local temperature increase can cause a total fusion of the selected cells or vesicles. Upon fusion, both the membrane and the cargos become completely mixed and we also show that the cells remain viable after fusion. The presented method has potential for single-cell targeted drug delivery and for the creation of hybrid cells.

  15. Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

    PubMed

    Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

    2017-09-11

    Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    SciTech Connect

    Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

    2014-12-12

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

  17. Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells

    PubMed Central

    Racanicchi, Serena; Maccherani, Chiara; Liberatore, Concetta; Billi, Monia; Gelmetti, Vania; Panigada, Maddalena; Rizzo, Giovanni; Nervi, Clara; Grignani, Francesco

    2005-01-01

    The AML1/ETO and PML/RARα leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARα and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARα–N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis. PMID:15729358

  18. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2009-07-01

    creates controlled flexion and extension in a mouse spine, so that spine fusions could be assessed using the same computer-assisted methods that are...reducing motion within the spine, we set up some experiments to look at flexion /tension under bending at specific angles. Briefly, three groups of...significant drop in the disc angle and in turn flexion of the spine. The reduction in flexion is consistent with what has been demonstrated in human

  19. Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles.

    PubMed

    Barona, Teresa; Byrne, Richard D; Pettitt, Trevor R; Wakelam, Michael J O; Larijani, Banafshe; Poccia, Dominic L

    2005-12-16

    Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.

  20. Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli.

    PubMed Central

    Kogoma, T; Farr, S B; Joyce, K M; Natvig, D O

    1988-01-01

    Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin. The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism. Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions. On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response. Introduction of oxyR, htpR, or recA mutations did not affect the induction. Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide. The third fusion strain showed no increased sensitivity to either agent. All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome. PMID:2838846

  1. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy

    PubMed Central

    Goh, Qingnian; Millay, Douglas P

    2017-01-01

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy. DOI: http://dx.doi.org/10.7554/eLife.20007.001 PMID:28186492

  2. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

    PubMed

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

  3. Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls

    PubMed Central

    Alvarez, Ashley Ann; Han, Sang Won; Toyota, Masatsugu; Brillada, Carla; Zheng, Jiameng; Gilroy, Simon

    2016-01-01

    Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 μm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception. PMID:27816929

  4. Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

    PubMed

    Permyakova, Natalia V; Zagorskaya, Alla A; Belavin, Pavel A; Uvarova, Elena A; Nosareva, Olesya V; Nesterov, Andrey E; Novikovskaya, Anna A; Zav'yalov, Evgeniy L; Moshkin, Mikhail P; Deineko, Elena V

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  5. Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process.

    PubMed

    Avitabile, Daniele; Crespi, Alessia; Brioschi, Chiara; Parente, Valeria; Toietta, Gabriele; Devanna, Paolo; Baruscotti, Mirko; Truffa, Silvia; Scavone, Angela; Rusconi, Francesca; Biondi, Andrea; D'Alessandra, Yuri; Vigna, Elisa; Difrancesco, Dario; Pesce, Maurizio; Capogrossi, Maurizio C; Barbuti, Andrea

    2011-05-01

    The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34

  6. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  7. Cell fusion mediates dramatic alterations in the actin cytoskeleton, focal adhesions, and E-cadherin in trophoblastic cells.

    PubMed

    Ishikawa, Atsuko; Omata, Waka; Ackerman, William E; Takeshita, Toshiyuki; Vandré, Dale D; Robinson, John M

    2014-04-01

    The syncytiotrophoblast of the human placenta is a unique epithelia structure with millions of nuclei sharing a common cytoplasm. The syncytiotrophoblast forms by cell-cell fusion of cytotrophoblasts (CTB), the mononuclear precursor cells. The trophoblastic BeWo cell line has been used as a surrogate for CTB since they can be induced to fuse, and subsequently display numerous syncytiotrophoblast differentiation markers following syncytial formation. In this study, we have focused on alterations in the cell-adhesion molecule E-cadherin, actin cytoskeleton, and focal adhesions following BeWo cell fusion, since these entities may be interrelated. There was a dramatic reorganization of the distribution of E-cadherin as well as a reduction in the amount of E-cadherin following cell fusion. Reorganization of the actin cytoskeleton was also observed, which was associated with a change in the globular actin (G-actin)/filamentous actin (F-actin) ratio. Concomitantly, the morphology of focal adhesions was altered, but this occurred without a corresponding change in the levels of focal adhesion marker proteins. Thus, extensive remodeling of the actin cytoskeleton and focal adhesions accompanies cell fusion and differentiation and appears related to alterations in E-cadherin in trophoblastic cells.

  8. HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

    PubMed

    Wang, Linya; Kim, Ja Yeon; Liu, Helene Minyi; Lai, Michael M C; Ou, Jing-Hsiung James

    2017-09-01

    Hepatitis C virus (HCV) induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7). Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER). Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

  9. Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

    PubMed Central

    Wu, Zhenyong; Auclair, Sarah M.; Bello, Oscar; Vennekate, Wensi; Dudzinski, Natasha R.; Krishnakumar, Shyam S.; Karatekin, Erdem

    2016-01-01

    The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability. PMID:27264104

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

    PubMed Central

    1994-01-01

    The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve". PMID:7806567

  11. Hot cell facility design for large fusion devices

    SciTech Connect

    Barrett, R.J.; Bussell, G.T.

    1985-01-01

    Large hot cell facilities will be necessary to support the operation of large fusion devices. The supporting hot cells will be needed to serve a variety of different functions and tasks, which include reactor component maintenance, tool and maintenance equipment repair, and preparation of radioactive material for shipment and disposal. This paper discusses hot cell facility functions, requirements, and design issues and techniques. Suggested solutions and examples are given.

  12. A cell attracting composite of lumbar fusion cage.

    PubMed

    Gunay, Busra; Hasirci, Nesrin; Hasirci, Vasif

    2017-06-01

    Lumbar fusion cages are devices used in spinal fusion procedures for disorders such as spondylosis and degenerative disc diseases that may occur due to age, trauma or genetic reasons. These devices are most frequently made of metals and polymers. The mechanical properties of such devices should be comparable to the bone to avoid stress shielding. Besides, cages should interact with the cells to prevent extrusion and achieve satisfactory fusion. In this study, poly(methylmethacrylate) (PMMA) and hydroxyapatite (HAp) were compounded to create products with HAp contents up to 40% (w/w), processed by hot melt extrusion and injection molded to produce composites with maximum polymer-mineral interaction. The morphology, interaction with the plates and rate of proliferation of human osteoblast-like (HOB) cells were studied in vitro. We learned that cells interact more with HAp when the HAp content is higher than 20%. Tensile and compressive properties of PMMA were significantly increased with increasing HAp content; from an elastic modulus (E) of 2.08 to 3.92 GPa in tension, and from 349 to 562 MPa in compression. High HAp content of the samples increased the roughness from 0.69 μm for pure PMMA to 1.35 μm for 40% (w/w) HAp loaded PMMA, increased cell proliferation and as a result the cells presented filopodia indicating a satisfactory level of interaction with the cage surface. Based on mechanical and in vitro studies, a HAp content of around 30% (w/w) was found to be appropriate for good cell adhesion and satisfactory mechanical properties for use in the construction of a fusion cage. It was concluded that when PMMA and HAp were compounded at an optimal value, a cage material with adequate mechanical properties and increased cell attachment can be obtained for use in spinal fusion applications.

  13. The Interaction of Mitochondrial Biogenesis and Fission/Fusion Mediated by PGC-1α Regulates Rotenone-Induced Dopaminergic Neurotoxicity.

    PubMed

    Peng, Kaige; Yang, Likui; Wang, Jian; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Cai, Ying; Cui, Zhihong; Ao, Lin; Liu, Jinyi; Zou, Zhongmin; Sai, Yan; Cao, Jia

    2017-07-01

    Parkinson's disease is a common neurodegenerative disease in the elderly, and mitochondrial defects underlie the pathogenesis of PD. Impairment of mitochondrial homeostasis results in reactive oxygen species formation, which in turn can potentiate the accumulation of dysfunctional mitochondria, forming a vicious cycle in the neuron. Mitochondrial fission/fusion and biogenesis play important roles in maintaining mitochondrial homeostasis. It has been reported that PGC-1α is a powerful transcription factor that is widely involved in the regulation of mitochondrial biogenesis, oxidative stress, and other processes. Therefore, we explored mitochondrial biogenesis, mitochondrial fission/fusion, and especially PGC-1α as the key point in the signaling mechanism of their interaction in rotenone-induced dopamine neurotoxicity. The results showed that mitochondrial number and mass were reduced significantly, accompanied by alterations in proteins known to regulate mitochondrial fission/fusion (MFN2, OPA1, Drp1, and Fis1) and mitochondrial biogenesis (PGC-1α and mtTFA). Further experiments proved that inhibition of mitochondrial fission or promotion of mitochondrial fusion has protective effects in rotenone-induced neurotoxicity and also promotes mitochondrial biogenesis. By establishing cell models of PGC-1α overexpression and reduced expression, we found that PGC-1α can regulate MFN2 and Drp1 protein expression and phosphorylation to influence mitochondrial fission/fusion. In summary, it can be concluded that PGC-1α-mediated cross talk between mitochondrial biogenesis and fission/fusion contributes to rotenone-induced dopaminergic neurodegeneration.

  14. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    PubMed

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Effects of polycystin‑1 N‑terminal fragment fusion protein on the proliferation and apoptosis of rat mesangial cells.

    PubMed

    Guan, Tianjun; Gao, Qing; Chen, Ping; Fu, Lili; Zhao, Haidan; Zou, Zhuying; Mei, Changlin

    2014-09-01

    Mesangial proliferative glomerulonephritis (MsPGN) is characterized by widespread mesangial cell proliferation and an accumulation of extracellular matrix (ECM) in the mesangial area. In a previous study we developed a polycystin‑1 N‑terminal fragment (PC‑1 NF) fusion protein that inhibits the proliferation of cyst‑lining epithelial cells in autosomal dominant polycystic kidney disease. In addition, the PC‑1 NF fusion protein arrests the cell cycle of cancer cells at the G0/G1 phase, inhibiting their proliferation. In the present study, the effect of the PC‑1 NF fusion protein on MsPGN was investigated. It was found that the PC‑1 NF fusion protein inhibited the proliferation of rat mesangial cells and induced G0/G1 phase arrest and apoptosis in vitro. PC‑1 NF fusion protein treatment also resulted in a decrease in mRNA expression levels of proliferating cell nuclear antigen, cyclin D1 and B‑cell lymphoma‑2 (Bcl‑2) and an increase in mRNA expression levels of Bcl‑2‑associated X protein (Bax) and p21Waf1. Furthermore, a decrease in Bcl‑2, c‑fos, c‑jun and protein kinase C‑α protein levels was observed, whereas Bax protein levels increased. Additionally, PC‑1 NF fusion protein induced ECM degradation and inhibited ECM expansion. The results also demonstrated that PC‑1 NF fusion protein treatment resulted in a decrease in type IV collagen and tissue inhibitor of metalloproteinase mRNA levels but an increase in matrix metalloproteinase 2 mRNA levels. In combination, these results suggest that the PC‑1 NF fusion protein inhibits proliferation, promotes apoptosis and induces ECM degradation in MsPGN rats. This study offers novel perspectives for the treatment of MsPGN.

  16. In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

    PubMed

    Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

    2012-10-12

    Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells

    PubMed Central

    Dittmar, Thomas; Zänker, Kurt S.

    2015-01-01

    The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that “accidental” tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells. PMID:26703575

  18. Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

    PubMed

    Geng, Yang; Zhou, Yan; Wu, Sai; Hu, Yabin; Lin, Kai; Wang, Yalin; Zheng, Zhongnan; Wu, Wei

    2017-01-01

    Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

  19. pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation.

    PubMed

    Ratnayake, Punsisi U; Sackett, Kelly; Nethercott, Matthew J; Weliky, David P

    2015-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region hypothesized to bind to the host cell membrane followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The present study focuses on the large gp41 ectodomain constructs "Hairpin" (HP) containing NHR+loop+CHR and "FP-Hairpin" (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH4 where the protein is positively-charged but do not induce fusion at pH7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. There are two kinetically distinct fusion processes at pH4: (1) a faster ~100 ms⁻¹ process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms⁻¹ process with extent strongly inversely correlated with this charge. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP's in an intermolecular antiparallel sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound proteins. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral p

  20. Cell fusion in the brain: two cells forward, one cell back.

    PubMed

    Kemp, Kevin; Wilkins, Alastair; Scolding, Neil

    2014-11-01

    Adult stem cell populations, notably those which reside in the bone marrow, have been shown to contribute to several neuronal cell types in the rodent and human brain. The observation that circulating bone marrow cells can migrate into the central nervous system and fuse with, in particular, cerebellar Purkinje cells has suggested, at least in part, a potential mechanism behind this process. Experimentally, the incidence of cell fusion in the brain is enhanced with age, radiation exposure, inflammation, chemotherapeutic drugs and even selective damage to the neurons themselves. The presence of cell fusion, shown by detection of increased bi-nucleated neurons, has also been described in a variety of human central nervous system diseases, including both multiple sclerosis and Alzheimer's disease. Accumulating evidence is therefore raising new questions into the biological significance of cell fusion, with the possibility that it represents an important means of cell-mediated neuroprotection or rescue of highly complex neurons that cannot be replaced in adult life. Here, we discuss the evidence behind this phenomenon in the rodent and human brain, with a focus on the subsequent research investigating the physiological mechanisms of cell fusion underlying this process. We also highlight how these studies offer new insights into endogenous neuronal repair, opening new exciting avenues for potential therapeutic interventions against neurodegeneration and brain injury.

  1. Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes

    PubMed Central

    2016-01-01

    Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and invivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivo applications. PMID:27725960

  2. Bone resorptive activity of human peripheral blood mononuclear cells after fusion with polyethylene glycol.

    PubMed

    Manrique, Edwin; Castillo, Luz M; Lazala, Oswaldo; Guerrero, Carlos A; Acosta, Orlando

    2017-03-01

    The bone remodeling process occurs through bone formation by osteoblasts and bone resorption by osteoclasts, a process involving the contribution of endocrine and nervous systems. The mechanisms associated to differentiation and proliferation of osteoclasts and osteoblasts are considered a potential therapeutic target for treating some erosive bone diseases. The aim of the present study is to explore the feasibility of generating active osteoclast-like cells from peripheral blood mononuclear cells (PBMCs) following polyethylene glycol (PEG)-induced fusion. PEG-fused PBMCs showed TRAP(+)-multinucleated cells and bone resorption activity, and were also positive for osteoclast markers such as carbonic anhydrase II, calcitonin receptor, vacuolar ATPase, and cathepsin K, when examined by reverse transcription-polymerase chain reaction, immunochemistry and Western blotting. TRAP expression and bone resorptive activity were higher in whole PEG-fused PBMCs than in separated T lymphocytes, B lymphocytes or monocytes. Both TRAP expression and bone resorptive activity were also higher in osteogenesis imperfecta patients compared to PEG-fused PBMCs from healthy individuals. PEG-induced fusion was more efficient in inducing TRAP and bone resorptive activities than macrophage colony-stimulating factor or dexamethasone treatment. Bone resorptive activity of PEG-fused PMBCs was inhibited by bisphosphonates. Evidence is provided that the use of PEG-based cell fusion is a straightforward and amenable method for studying human osteoclast differentiation and testing new therapeutic strategies.

  3. RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion

    PubMed Central

    Smurova, Ksenia; Podbilewicz, Benjamin

    2016-01-01

    Summary Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen), which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion. PMID:26854231

  4. Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

    PubMed Central

    Wang, Yucai; Liu, Yunyan; Zheng, Lianhe

    2014-01-01

    Background Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown. Methods Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared. Results Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction. Conclusions The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy. PMID:24466232

  5. Heme-mediated apoptosis and fusion damage in BeWo trophoblast cells

    PubMed Central

    Liu, Mingli; Hassana, Salifu; Stiles, Jonathan K.

    2016-01-01

    Placental malaria (PM) is a complication associated with malaria infection during pregnancy that often leads to abortion, premature delivery, intrauterine growth restriction and low birth weight. Increased levels of circulating free heme, a by-product of Plasmodium-damaged erythrocytes, is a major contributor to inflammation, tissue damage and loss of blood brain barrier integrity associated with fatal experimental cerebral malaria. However, the role of heme in PM remains unknown. Proliferation and apoptosis of trophoblasts and fusion of the mononucleated state to the syncytial state are of major importance to a successful pregnancy. In the present study, we examined the effects of heme on the viability and fusion of a trophoblast-derived cell line (BeWo). Results indicate that heme induces apoptosis in BeWo cells by activation of the STAT3/caspase-3/PARP signaling pathway. In the presence of forskolin, which triggers trophoblast fusion, heme inhibits BeWo cell fusion through activation of STAT3. Understanding the effects of free plasma heme in pregnant women either due to malaria, sickle cell disease or other hemolytic diseases, will enable identification of high-risk women and may lead to discovery of new drug targets against associated adverse pregnancy outcome. PMID:27796349

  6. Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells.

    PubMed

    Lizunov, Vladimir A; Lee, Jo-Ping; Skarulis, Monica C; Zimmerberg, Joshua; Cushman, Samuel W; Stenkula, Karin G

    2013-09-01

    Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (Si) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject Si, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing Si, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with Si and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor Si, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction.

  7. Cell-fusion method to visualize interphase nuclear pore formation.

    PubMed

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

    In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion.

    PubMed

    Gordón-Alonso, Mónica; Yañez-Mó, María; Barreiro, Olga; Alvarez, Susana; Muñoz-Fernández, M Angeles; Valenzuela-Fernández, Agustín; Sánchez-Madrid, Francisco

    2006-10-15

    Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

  9. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

    PubMed

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S

    2017-03-15

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular

  10. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  11. TALEN-Induced Translocations in Human Cells.

    PubMed

    Piganeau, Marion; Renouf, Benjamin; Ghezraoui, Hind; Brunet, Erika

    2016-01-01

    Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

  12. Role and organization of the actin cytoskeleton during cell-cell fusion.

    PubMed

    Martin, Sophie G

    2016-12-01

    Cell-cell fusion is a ubiquitous process that underlies fertilization and development of eukaryotes. This process requires fusogenic machineries to promote plasma membrane merging, and also relies on the organization of dedicated sub-cortical cytoskeletal assemblies. This review describes the role of actin structures, so called actin fusion foci, essential for the fusion of two distinct cell types: Drosophila myoblast cells, which fuse to form myotubes, and sexually differentiated cells of the fission yeast Schizosaccharomyces pombe, which fuse to form a zygote. I describe the respective composition and organization of the two structures, discuss their proposed role in promoting plasma membrane apposition, and consider the universality of similar structures for cell-cell fusion.

  13. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein.

    PubMed

    Nervi, C; Ferrara, F F; Fanelli, M; Rippo, M R; Tomassini, B; Ferrucci, P F; Ruthardt, M; Gelmetti, V; Gambacorti-Passerini, C; Diverio, D; Grignani, F; Pelicci, P G; Testi, R

    1998-10-01

    All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.

  14. Temperature dependence of calcium-induced fusion of sonicated phosphatidylserine vesicles.

    PubMed Central

    Sun, S T; Day, E P; Ho, J T

    1978-01-01

    We have measured the temperature dependence calcium-induced fusion of sonicated phosphatidylserine vesicles. The vesicles were incubated in the presence of calcium at a specified temperature until the resulting aggregation or fusion process had gone to completion. EDTA was then added and the resulting final size of the vesicle population was measured by using dynamic light scattering. This final size was plotted against incubation temperature to show the temperature dependence of calcium-induced fusion. This curve has a peak near 11 degrees C which may be associated with the phase transition of the sonicated phosphatidylserine vesicles in the presence of calcium prior to the aggregation or fusion process. PMID:279918

  15. Cell Therapy to Obtain Spinal Fusion

    DTIC Science & Technology

    2008-07-01

    hydrogel materials. We are also currently developing a near infrared dye IR800 that will enter cells and bind to a peptide moiety known as halo tag...Promega Corp). We believe the near infrared will provide the greatest sensitivity. However, we are including preliminary data comparing three...injection. We propose that introduction of the near infrared dye will allow us to detect as few as 100 cells. We propose to complete these

  16. Hemi-fused structure mediates and controls fusion and fission in live cells

    PubMed Central

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J.; Krystofiak, Evan S.; Villarreal, Seth A.; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-01-01

    Membrane fusion and fission are vital to eukaryotes’ life1–5. For three decades, it has been proposed that fusion is mediated by fusion between proximal leaflets of two bilayers (hemi-fusion) that produces a hemi-fused structure, followed by fusion between distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission1, 4, 6–10. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion/hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed2, 11–15. Using confocal and super-resolution STED microscopy, we observed the hemi-fused Ω-shaped structure for the first time in live cells, neuroendocrine chromaffin cells and pancreatic β-cells. This structure was generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, its transition to full fusion or fission was determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and was surprisingly slow (seconds to tens of seconds) in a significant fraction of the events. These results provide key missing evidence over the past three decades proving the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion/fission, as fusion and fission mechanisms compete to determine its transition to fusion or fission. PMID:27309816

  17. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  18. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  19. Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a.

    PubMed

    Tang, Chao; Tang, Lanfang; Wu, Xiaokai; Xiong, Wenyi; Ruan, Hongfeng; Hussain, Musaddique; Wu, Junsong; Zou, Chaochun; Wu, Ximei

    2016-03-11

    Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance.

  20. Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a*

    PubMed Central

    Tang, Chao; Tang, Lanfang; Wu, Xiaokai; Xiong, Wenyi; Ruan, Hongfeng; Hussain, Musaddique; Wu, Junsong; Zou, Chaochun; Wu, Ximei

    2016-01-01

    Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance. PMID:26769961

  1. A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization.

    PubMed

    Potala, Sirisha; Verma, Rama Shanker

    2010-11-01

    Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.

  2. Two Photon Absorption Laser Induced Fluorescence for Fusion Class Plasmas

    NASA Astrophysics Data System (ADS)

    Elliott, Drew B.

    Neutral hydrogen particles play an important role in many fusion systems. The edge region of fusion plasmas is strongly influenced by these neutral particles and is of growing importance because of the challenges of plasma material interaction. A two photon absorption laser induced fluorescence diagnostic at West Virginia University has been constructed to measure the local density and velocity distribution of these neutral particles. The diagnostic measures the ground state of hydrogen isotopes by way of two photon absorption from the 1s to 3d state and subsequent single photon emission to the 2 p state. These measurements are absolutely calibrated by comparing the integrated emission spectra to that of a measurement performed on a known density of calibration gas and knowing the relative absorption cross sections for the two species. Measurements were performed on deuterium atoms in the Helicity Injected Torus with Steady Induction 3 and calibrated using the standard krypton calibration scheme. Measured neutral densities were well below predicted values and the measurement process identified a flaw in the krypton calibration scheme. A new calibration scheme using xenon gas was developed to eliminate any possibility of chromatic aberration through refractive optics. This new xenon calibration scheme required measurement of the relative absorption cross section between the 5p6 to 4p 57f to 5p55 d Xe scheme and the 4p6 to 4 p55p to 4p 55s Kr scheme, then comparison of the Xe to Kr relative cross section to the Kr to H relative cross section to determine the overall Xe to H relative absorption cross section. Doppler free two photon absorption laser induced fluorescence measurements were also performed on the compact helicon for waves and instabilities experiment (CHEWIE), for hydrogen, deuterium, and krypton neutrals. The Doppler free technique increased signal intensity and narrowed the measured spectral width of the absorption line. The Doppler free technique

  3. Enhanced MyoD-Induced Transdifferentiation to a Myogenic Lineage by Fusion to a Potent Transactivation Domain

    PubMed Central

    2015-01-01

    Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine. PMID:25494287

  4. Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

    PubMed

    Kabadi, Ami M; Thakore, Pratiksha I; Vockley, Christopher M; Ousterout, David G; Gibson, Tyler M; Guilak, Farshid; Reddy, Timothy E; Gersbach, Charles A

    2015-06-19

    Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

  5. An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

    PubMed

    Bird, R Curtis; Deinnocentes, Patricia; Church Bird, Allison E; van Ginkel, Frederik W; Lindquist, Joni; Smith, Bruce F

    2011-01-01

    Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

  6. Hemi-fused structure mediates and controls fusion and fission in live cells.

    PubMed

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

  7. Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

    PubMed Central

    Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

    2013-01-01

    The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

  8. Characterization of functionally active interleukin-18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shien, Jui Hung; Shen, Pin Chun; Lee, Long Huw

    2016-05-01

    The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL-18a cell line that stably expresses the fusion protein chIL-18 was constructed and the enhanced green fluorescence protein connected through a (G4 S)3 linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL-18a cells (1 × 10(5) ) were inoculated in 60-mm culture dishes containing 5 mL of media to achieve 50%-60% confluence and were cultured in the presence of the cycle-specific inhibitors 10058-F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058-F4, aphidicolin, and colchicine, respectively, the aphidicolin-induced single cells showed higher fusion protein levels than did the 10058-F4- or colchicine-induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin-treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN-γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581-591, 2016.

  9. FUSION-COMPETENT STATE INDUCED BY A C-TERMINAL HIV-1 FUSION PEPTIDE IN CHOLESTEROL-RICH MEMBRANES

    PubMed Central

    Apellániz, Beatriz; Nieva, José L.

    2015-01-01

    The replicative cycle of the Human Immunodeficiency Virus type-1 begins after fusion of the viral and target-cell membranes. The envelope glycoprotein gp41 transmembrane subunit contains conserved hydrophobic domains that engage and perturb the merging lipid bilayers. In this work, we have characterized the fusion-committed state generated in vesicles by CpreTM, a synthetic peptide derived from the sequence connecting the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of gp41. Pre-loading cholesterol-rich vesicles with CpreTM rendered them competent for subsequent lipid-mixing with fluorescently-labeled target vesicles. Highlighting the physiological relevance of the lasting fusion-competent state, the broadly neutralizing antibody 4E10 bound to the CpreTM-primed vesicles and inhibited lipid-mixing. Heterotypic fusion assays disclosed dependence on the lipid composition of the vesicles that acted either as virus or cell membrane surrogates. Lipid-mixing exhibited above all a critical dependence on the cholesterol content in those experiments. We infer that the fusion-competent state described herein resembles bona-fide perturbations generated by the pre-hairpin MPER-TMD connection within the viral membrane. PMID:25617671

  10. Cell Fusion as a Cofactor in Prostate Cancer Metastasis

    DTIC Science & Technology

    2010-01-01

    fusion. However, our findings indicated a different and unexpected to us mechanism. We found that PC-3 released one or more infectious viruses that...and MLV are, we could not identify the virus secreted by PC-3 cells unambiguously. While were we conducting our studies , several reports questioned...vesicular stomatitis virus (VSV), to be reversibly activated by a brief incubation in mildly acidic medium. The advantages of this method are that it is not

  11. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    SciTech Connect

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  12. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  13. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    SciTech Connect

    Gambhir,; Sanjiv, Pritha; Ray,

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  14. Antitumor response elicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells.

    PubMed

    Wahlsten, J L; Mills, C D; Ramakrishnan, S

    1998-12-15

    Superantigens stimulate T cells bearing certain TCR beta-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST(88-194)-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST(88-194)-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST(88-194)-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST(88-194)-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.

  15. Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

    PubMed

    Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

    2006-02-01

    The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

  16. Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming

    PubMed Central

    Kouris, Nicholas A.; Schaefer, Jeremy A.; Hatta, Masato; Freeman, Brian T.; Kamp, Timothy J.; Kawaoka, Yoshihiro; Ogle, Brenda M.

    2012-01-01

    Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies. PMID:22701126

  17. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    SciTech Connect

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  18. [Experimental study on the immune response of fusion tumor vaccine of HepG2 and dendritic cells in vitro].

    PubMed

    Pang, Y B; Cui, B Y; He, J; Huang, X P; Liang, W; Li, L Q; Luo, X L

    2017-02-21

    Objective: To estimate the immune response of HepG2/dendritic cell (DC) fusion cells vaccines against HepG2 cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation.Then DC were obtain from PBMCs by culturing in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days.DC and HepG2 fusion cells were induced by polythyleneglycol (PEG). The fusion cells were examined under fluorescence microscope by labeling DCs and HepG2 with green and red fluorescein, respectively, and then the fusion rates were analyzed by flow cytometry.The capacity of fusion cells to secrete interleukin (IL)-12 and stimulate the proliferation of T lymphocyte was assessed by ELISA and Flow cytometry, respectively.ELISPOT was used to assess the interferon gamma (IFN-γ) produced by cytotoxicity T lymphocyte (CTL), and the specific killing ability of fusion cells induce-CTL targeting HepG2 was estimated. Results: The fusion rate of HepG2/DC was 54.5%, and the fusion cells expressed a higher levels of DC mature marker CD80 and costimulatory molecules CD83, CD86 and MHC-Ⅰ, MHC-Ⅱ molecules HLA-ABC and HLA-DR than those in immature DCs (P<0.01). HepG2/DC showed a greater capacity to secrete high level of IL-12 (P<0.05) and activate proliferation of lymphocytes in vitro, as compared with DCs alone and DCs mix HepG2 (P<0.01). The HepG2/DC -activated CTL generated higher IFN-γ level and had a specific killing ability against HepG2 cells at the effecter/target ratio 30∶1 (31.4%±2.4%) and 100∶1 (57.6%±7.3%) (P<0.01). Conclusions: HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells.It might be a promising strategy of immunotherapy for HCC.

  19. Search for systematic behavior of incomplete-fusion probability and complete-fusion suppression induced by {sup 9}Be on different targets

    SciTech Connect

    Gomes, P. R. S.; Linares, R.; Lubian, J.; Lopes, C. C.; Cardozo, E. N.; Pereira, B. H. F.

    2011-07-15

    We present a trial to obtain a systematic behavior of the results available in the literature on the complete and incomplete fusion induced by the weakly bound projectile {sup 9}Be on targets with different masses and/or charges. We stress that although the incomplete-fusion probability and the complete-fusion suppression are very closely related quantities, the first is an experimental value whereas the later is model dependent. A trend of systematic behavior for the incomplete-fusion probability as a function of the target charge is achieved, but not for the complete-fusion suppression.

  20. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    PubMed

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

    NASA Technical Reports Server (NTRS)

    Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  2. The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

    NASA Technical Reports Server (NTRS)

    Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  3. The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

    NASA Technical Reports Server (NTRS)

    Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  4. Vaccination with Dendritic Cell Myeloma Fusions in Conjunction With Stem Cell Transplantation and PD1 Blockade

    DTIC Science & Technology

    2010-05-01

    glycol. Mean expression of PDL- 1 was 97% on DCs (n=5, Figure 1), and 45% on patient derived myeloma cells (n=6, Figure 1). In addition, mean... expression of PDL-1 was 90% on DC/ myeloma fusions (n=2, Figure 2), suggesting this pathway may provide an inhibitory signal that blunts fusion mediated...immunologic response. Expression of PD-1 on T cells isolated from patients with multiple myeloma : In infectious disease models, upregulation of T

  5. Fusion and metabolism of plant cells as affected by microgravity.

    PubMed

    Hampp, R; Hoffmann, E; Schönherr, K; Johann, P; De Filippis, L

    1997-01-01

    Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress

  6. pH-Dependent Vesicle Fusion Induced by the Ectodomain of the Human Immunodeficiency Virus Membrane Fusion Protein gp41: Two Kinetically Distinct Processes and Fully-Membrane-Associated gp41 with Predominant β Sheet Fusion Peptide Conformation

    PubMed Central

    Ratnayake, Punsisi U.; Sackett, Kelly; Nethercott, Matthew J.; Weliky, David P.

    2014-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region

  7. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    PubMed

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  8. Calculations of Proton Emission Cross Sections in Deuteron Induced Reactions of Some Fusion Structural Materials

    NASA Astrophysics Data System (ADS)

    Yiğit, M.; Tel, E.; Tanır, G.

    2013-06-01

    The growing demands for energy consumption have led to the increase of the research and development activities on new energy sources. Fusion energy has the highest potential to become a very safe, clean and abundant energy source for the future. To get energy from fusion are needed for development of fusion reactor technology. Particularly, the design and development of international facilities as International Thermonuclear Experimental Reactor and International Fusion Material Irradiation Facility requires for the cross-section data of deuteron induced reactions. Moreover, the selection of fusion structural materials are an indispensable component for this technology. Therefore, the cross-section data of deuteron induced reactions on fusion structural materials are of great importance for development of fusion reactor technology. In this study, reaction model calculations of the cross sections of deuteron induced reactions on structural fusion materials such as 27Al, 59Co, 55Mn, 50Cr, 54Cr, 64Ni, 109Ag, 184W and 186W have been carried out for incident energies up to 50 MeV. In these calculations, the pre-equilibrium and equilibrium effects for ( d, p) stripping reactions have been investigated. The pre-equilibrium calculations involve the new evaluated the geometry dependent hybrid model and hybrid model. Equilibrium effects are calculated according to the Weisskopf-Ewing model. In the calculations the program code ALICE/ASH was used. The calculated results are discussed and compared with the experimental data taken from the literature.

  9. Pronuclear fusion failure: an alternate conjugational pathway in Tetrahymena thermophila, induced by vinblastine

    SciTech Connect

    Hamilton, E.P.; Suhr-Jessen, P.B.; Orias, E.

    1988-04-01

    Vinblastine is shown to induce pronuclear fusion failure in conjugating Tetrahymena thermophila. In this alternate conjugational pathway gametic pronuclei are exchanged between conjugants but do not fuse. Each pronucleus undergoes one mitotic division to produce a new macro- and micronucleus. Genetic consequences of pronuclear fusion failure include the following: (1) the progeny are whole genome homozygotes with nuclei derived from single meiotic products, and (2) half of the progeny are heterokaryons with micro- and macronuclei of different genetic origins. These facts make this process extremely useful in strain construction and mutant isolation. The induction of pronuclear fusion failure by vinblastine suggests that microtubules play an essential role in pronuclear fusion. In this paper the authors present autoradiographic and genetic evidence that vinblastine induces pronuclear fusion failure in T. thermophila.

  10. Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion.

    PubMed

    Nakane, Shuhei; Matsuda, Zene

    2015-01-01

    Fusion between viral and cellular membranes is the essential first step in infection of enveloped viruses. This step is mediated by viral envelope glycoproteins (Env) that recognize cellular receptors. The membrane fusion between the effector cells expressing viral Env and the target cells expressing its receptors can be monitored by several methods. We have recently developed a pair of chimeric reporter protein composed of split Renilla luciferase (RL) and split GFP. We named this reporter dual split protein (DSP), since it recovers both RL and GFP activities upon self reassociation. By using DSP, pore formation and content mixing between the effector and target cells can be monitored upon the recovery of RL and GFP activities after the membrane fusion. This quick assay provides quantitative as well as spatial information about membrane fusion mediated by viral Env.

  11. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    SciTech Connect

    Karentz, D.; Cleaver, J.E.

    1986-10-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.

  12. Fusion of Mouse-Mouse Cells to Produce Hybridomas Secreting Monoclonal Antibody,

    DTIC Science & Technology

    the plasmacytoma cells for fusion, the fusion method, and the method of taking samples for identification of colonies secreting monoclonal antibody...The plasmacytoma cell line used is designated SP2/0 ag14. This continuous cell line is a nonimmunoglobulin secreting mouse hybridoma cell line

  13. Two modes of lytic granule fusion during degranulation by natural killer cells.

    PubMed

    Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer, John A; Long, Eric O

    2011-08-01

    Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

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

    PubMed Central

    Merlini, Laura

    2016-01-01

    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. PMID:27798845

  15. Laser-assisted cell fusion and cytoplast transfer in early mammalian embryos

    NASA Astrophysics Data System (ADS)

    Clement-Sengewald, Annette; Schutze, Karin; Heinze, A.; Palma, G. A.; Poesl, H.; Brem, G.

    1993-07-01

    A UV-laser microbeam was successfully used to induce fusion of early embryonic cells. The developmental capacity of the laser-fused cells was examined using in vitro culture methods. Blastomeres within mouse two-cell embryos were fused with 3 - 10 subsequent laser pulses in order to produce tetraploid embryos. Thirty-one percent of the laser treated embryos fused and 10% of those developed to the morula or blastocyst stage. With 1 - 10 successive laser pulses cattle oocytes were fused with cytoplasts. Thirty-six percent of the laser treated cells fused and 10% of those cleaved to the 6- and 8-cell stage. These preliminary results indicate that a UV- laser microbeam combined with an optical tweezers may facilitate the manipulation of embryonic cells and can be a helpful tool in polyploidy studies and in cytoplasmic transfer experiments.

  16. MiR-106b-mediated Mfn2 suppression is critical for PKM2 induced mitochondrial fusion

    PubMed Central

    Wu, Haili; Li, Zhuoyu; Wang, Yingying; Yang, Peng; Li, Zongrui; Li, Hanqing; Wu, Changxin

    2016-01-01

    Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is a characteristic of glucose metabolism in cancer cells. PKM2 is reported to imply an important role in glycolysis. However, whether and how PKM2 can cause mitochondrial dysfunction, then subsequently forcing cancer cells using glycolysis instead of oxidation phosphorylation is poorly understood. Here we reported that overexpression of PKM2 disrupted mitochondrial dynamics by enhancing fusion. And PKM2 overexpression increased the expression of fusion protein Mfn2. Simultaneously, PKM2 overexpression induced mitochondrial dysfunctions shown by the decreased ATP level and increased mitochondrial DNA (mtDNA) copy number. Reduction of Mfn2 expression by siRNA attenuated the PKM2-enhanced mitochondrial fusion and restored the functions. Quantitative and morphological analyses showed that the expression of microRNA-106b (miR-106b) was decreased in the PKM2 overexpressed cells, and the reduction of Mfn2 expression and the recovery of mitochondrial functions were induced by the treatment of miR-106b mimics, demonstrating that miR-106b played important roles in the down-regulation of Mfn2 expression and the PKM2 mediation of mitochondrial fusion. Clinical investigation was performed and results showed that the higher expression levels of PKM2 corresponded with the higher expression levels of Mfn2 in breast cancer tissues by comparison of their expression in adjacent normal tissues. Taken together, our data demonstrate that the overexpression of PKM2 and Mfn2 causes mitochondrial dysfunction via enhancing mitochondrial fusion and miR-106b play crucial roles in PKM2 mediated mitochondrial function through its regulation of Mfn2 expression, which provides new insights into the molecular mechanisms underlying glycolysis and oxidative phosphorylation. PMID:27822413

  17. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    PubMed

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-07

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.

  18. High perfluorooctanoic acid exposure induces autophagy blockage and disturbs intracellular vesicle fusion in the liver.

    PubMed

    Yan, Shengmin; Zhang, Hongxia; Guo, Xuejiang; Wang, Jianshe; Dai, Jiayin

    2017-01-01

    Perfluorooctanoic acid (PFOA) has been shown to cause hepatotoxicity and other toxicological effects. Though PPARα activation by PFOA in the liver has been well accepted as an important mechanism of PFOA-induced hepatotoxicity, several pieces of evidence have shown that the hepatotoxic effects of PFOA may not be fully explained by PPARα activation. In this study, we observed autophagosome accumulation in mouse livers as well as HepG2 cells after PFOA exposure. Further in vitro study revealed that the accumulation of autophagosomes was not caused by autophagic flux stimulation. In addition, we observed that PFOA exposure affected the proteolytic activity of HepG2 cells while significant dysfunction of lysosomes was not detected. Quantitative proteomic analysis of crude lysosomal fractions from HepG2 cells treated with PFOA revealed that 54 differentially expressed proteins were related to autophagy or vesicular trafficking and fusion. The proteomic results were further validated in the cells in vitro and livers in vivo after PFOA exposure, which implied potential dysfunction at the late stage of autophagy. However, in HepG2 cells, it seemed that further inhibition of autophagy did not significantly alter the effects of PFOA on cell viability. Although these findings demonstrate that PFOA blocked autophagy and disturbed intracellular vesicle fusion in the liver, the changes in autophagy were observed only at high cytotoxic concentrations of PFOA, suggesting that autophagy may not be a primary target or mode of toxicity. Furthermore, since altered liver autophagy was not observed at concentrations of PFOA associated with human exposures, the relevance of these findings must be questioned.

  19. The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

    PubMed Central

    Sánchez-Migallón, M P; Aranda, F J; Gómez-Fernández, J C

    1995-01-01

    We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed. PMID:7696508

  20. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    PubMed

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  1. Direct transfer of learned behaviour via cell fusion in non-neural organisms.

    PubMed

    Vogel, David; Dussutour, Audrey

    2016-12-28

    Cell fusion is a fundamental phenomenon observed in all eukaryotes. Cells can exchange resources such as molecules or organelles during fusion. In this paper, we ask whether a cell can also transfer an adaptive response to a fusion partner. We addressed this question in the unicellular slime mould Physarum polycephalum, in which cell-cell fusion is extremely common. Slime moulds are capable of habituation, a simple form of learning, when repeatedly exposed to an innocuous repellent, despite lacking neurons and comprising only a single cell. In this paper, we present a set of experiments demonstrating that slime moulds habituated to a repellent can transfer this adaptive response by cell fusion to individuals that have never encountered the repellent. In addition, we show that a slime mould resulting from the fusion of a minority of habituated slime moulds and a majority of unhabituated ones still shows an adaptive response to the repellent. Finally, we further reveal that fusion must last a certain time to ensure an effective transfer of the behavioural adaptation between slime moulds. Our results provide strong experimental evidence that slime moulds exhibit transfer of learned behaviour during cell fusion and raise the possibility that similar phenomena may occur in other cell-cell fusion systems. © 2016 The Author(s).

  2. Functional characterisation of different MLL fusion proteins by using inducible Sleeping Beauty vectors.

    PubMed

    Wächter, K; Kowarz, E; Marschalek, R

    2014-10-01

    Our focus is the identification, characterisation and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  3. Investigation of contribution of incomplete fusion in the total fusion process induced by 9Be on 181Ta target at near barrier energies

    NASA Astrophysics Data System (ADS)

    Kharab, Rajesh; Chahal, Rajiv; Kumar, Rajiv

    2016-02-01

    We have studied the relative contribution of incomplete fusion (ICF) and complete fusion (CF) in total fusion (TF) induced by 9Be on 181Ta target at energies in the vicinity of Coulomb barrier using classical dynamical model and Wong's formula in conjugation with energy dependent Woods-Saxon formula. It is found that at above barrier energies ICF contributes almost 30% in TF while at energies below the barrier qualitatively its contribution is much more than thirty percent.

  4. Rvs161p Interacts with Fus2p to Promote Cell Fusion in Saccharomyces cerevisiae

    PubMed Central

    Brizzio, Valeria; Gammie, Alison E.; Rose, Mark D.

    1998-01-01

    FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion. PMID:9566960

  5. Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells

    PubMed Central

    Shadrin, Ilya Y.; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

    2015-01-01

    Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca2+ indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25–40% of hMSCs (from 4 independent donors) acquired periodic Ca2+ transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca2+ current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca2+ channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy. PMID:26159124

  6. Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

    PubMed

    Shadrin, Ilya Y; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

    2015-07-10

    Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

  7. Fusion of dendritic cells and CD34+CD38- acute myeloid leukemia (AML) cells potentiates targeting AML-initiating cells by specific CTL induction.

    PubMed

    Lei, Zhang; Zhang, Gui-Mei; Hong, Mei; Feng, Zuo-Hua; Huang, Bo

    2009-05-01

    Distinct leukemia-initiating cells (L-ICs) represent a critical target for therapeutic intervention of acute myeloid leukemia (AML). A potential strategy to eradicate L-ICs is to generate L-IC-specific cytotoxic T lymphocytes (CTLs). However, owing to rarity and immortality of L-ICs, it is difficult for antigen-presenting cells to capture L-ICs for specific antigen presentation. Here, we report a novel approach by fusing allogeneic dendritic cells (DCs) and CD34CD38 AML progenitor cells, through which specific CTLs were effectively induced, leading to the cytolysis to AML-initiating cells. Fusion of either DC/CD34CD38 AML cell or DC/CD34 AML cell could effectively induce the proliferation and activation of CTLs. However, only the former CTLs could effectively attack AML progenitor cells, and result in the unkilled progenitor/initiating cells losing the abilities of active proliferation in vitro and engraftment in NOD-SCID mice. These findings suggest that AML progenitor/initiating cell-specific CTLs may be generated based on allogeneic DC/progenitor cell fusion strategy; the induced CTLs may potentially eradicate AML by targeting L-ICs directly or indirectly.

  8. Identification and Characterization of Genes Required for Cell-to-Cell Fusion in Neurospora crassa ▿ †

    PubMed Central

    Fu, Ci; Iyer, Priyadarshini; Herkal, Amrita; Abdullah, Julia; Stout, Angela; Free, Stephen J.

    2011-01-01

    A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the Neurospora crassa single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (mik-1, mek-1, mak-1, nrc-1, mek-2, mak-2, rac-1, pp2A, so/ham-1, ham-2, ham-3, ham-5, ham-9, and mob3). The screening experiments also identified four transcription factors that are required for cell fusion (adv-1, ada-3, rco-1, and snf5). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (amph-1, ham-10, pkr1). Three of the genes identified by the screening procedure, ham-6, ham-7, and ham-8, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion. PMID:21666072

  9. Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.

    PubMed

    Yao, Fei; Kausalya, Jaya P; Sia, Yee Yen; Teo, Audrey S M; Lee, Wah Heng; Ong, Alicia G M; Zhang, Zhenshui; Tan, Joanna H J; Li, Guoliang; Bertrand, Denis; Liu, Xingliang; Poh, Huay Mei; Guan, Peiyong; Zhu, Feng; Pathiraja, Thushangi Nadeera; Ariyaratne, Pramila N; Rao, Jaideepraj; Woo, Xing Yi; Cai, Shaojiang; Mulawadi, Fabianus H; Poh, Wan Ting; Veeravalli, Lavanya; Chan, Chee Seng; Lim, Seong Soo; Leong, See Ting; Neo, Say Chuan; Choi, Poh Sum D; Chew, Elaine G Y; Nagarajan, Niranjan; Jacques, Pierre-Étienne; So, Jimmy B Y; Ruan, Xiaoan; Yeoh, Khay Guan; Tan, Patrick; Sung, Wing-Kin; Hunziker, Walter; Ruan, Yijun; Hillmer, Axel M

    2015-07-14

    Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed.

  10. Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

    SciTech Connect

    Wu Liguo; Hutt-Fletcher, Lindsey M. . E-mail: lhuttf@lsuhsc.edu

    2007-06-20

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

  11. Mesenchymal stem cells expressing baculovirus-engineered BMP-2 and VEGF enhance posterolateral spine fusion in a rabbit model.

    PubMed

    Fu, Tsai-Sheng; Chang, Yu-Han; Wong, Chak-Bor; Wang, I-Chun; Tsai, Tsung-Ting; Lai, Po-Liang; Chen, Lih-Huei; Chen, Wen-Jer

    2015-09-01

    were significantly greater in the MSC/Bac/TCP group. The results indicate the potential of using baculovirus as a vector for gene/cell therapy approaches to improve bone healing and support the feasibility of using allogeneic MSCs for inducing bone formation and intertransverse process fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The Problem of Colliding Networks and its Relation to Cell Fusion and Cancer

    PubMed Central

    Koulakov, Alexei A.; Lazebnik, Yuri

    2012-01-01

    Cell fusion, a process that merges two or more cells into one, is required for normal development and has been explored as a tool for stem cell therapy. It has also been proposed that cell fusion causes cancer and contributes to its progression. These functions rely on a poorly understood ability of cell fusion to create new cell types. We suggest that this ability can be understood by considering cells as attractor networks whose basic property is to adopt a set of distinct, stable, self-maintaining states called attractors. According to this view, fusion of two cell types is a collision of two networks that have adopted distinct attractors. To learn how these networks reach a consensus, we model cell fusion computationally. To do so, we simulate patterns of gene activities using a formalism developed to simulate patterns of memory in neural networks. We find that the hybrid networks can assume attractors that are unrelated to parental attractors, implying that cell fusion can create new cell types by nearly instantaneously moving cells between attractors. We also show that hybrid networks are prone to assume spurious attractors, which are emergent and sporadic network states. This finding means that cell fusion can produce abnormal cell types, including cancerous types, by placing cells into normally inaccessible spurious states. Finally, we suggest that the problem of colliding networks has general significance in many processes represented by attractor networks, including biological, social, and political phenomena. PMID:23199929

  13. Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

    PubMed

    Matsubara, Daisuke; Kanai, Yoshihiko; Ishikawa, Shumpei; Ohara, Shiori; Yoshimoto, Taichiro; Sakatani, Takashi; Oguni, Sachiko; Tamura, Tomoko; Kataoka, Hiroaki; Endo, Shunsuke; Murakami, Yoshinori; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro

    2012-12-01

    Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

  14. `Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

    NASA Astrophysics Data System (ADS)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

    Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (`Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called `full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of `full fusion'.

  15. 'Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells.

    PubMed

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

    Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering ('Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called 'full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of 'full fusion'.

  16. Microscopic dynamics simulations of heavy-ion fusion reactions induced by neutron-rich nuclei

    NASA Astrophysics Data System (ADS)

    Wang, Ning; Ou, Li; Zhang, Yingxun; Li, Zhuxia

    2014-06-01

    The heavy-ion fusion reactions induced by neutron-rich nuclei are investigated with the improved quantum molecular dynamics (ImQMD) model. With a subtle consideration of the neutron skin thickness of nuclei and the symmetry potential, the stability of nuclei and the fusion excitation functions of heavy-ion fusion reactions O16 + Ge76, O16 + Sm154, Ca40 + Zr96, and Sn132 + Ca40 are systematically studied. The fusion cross sections of these reactions at energies around the Coulomb barrier can be well reproduced by using the ImQMD model. The corresponding slope parameter of the symmetry energy adopted in the calculations is L ≈78 MeV and the surface energy coefficient is gsur=18±1.5 MeV fm2. In addition, it is found that the surface-symmetry term significantly influences the fusion cross sections of neutron-rich fusion systems. For sub-barrier fusion, the dynamical fluctuations in the densities of the reaction partners and the enhanced surface diffuseness at neck side result in the lowering of the fusion barrier.

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

    PubMed

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    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. The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic

  18. Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion

    PubMed Central

    Kato, Kagayaki; Dong, Bo; Wada, Housei; Tanaka-Matakatsu, Miho; Yagi, Yoshimasa; Hayashi, Shigeo

    2016-01-01

    Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. We show that as adhesion sites form between fusion cells, myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. We demonstrate that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. We also show that retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. We propose that microtubule bundles connecting these cell–cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion. PMID:27067650

  19. DC-SIGN Facilitates Fusion of Dendritic Cells with Human T-Cell Leukemia Virus Type 1-Infected Cells

    PubMed Central

    Ceccaldi, Pierre-Emmanuel; Delebecque, Frédéric; Prevost, Marie-Christine; Moris, Arnaud; Abastado, Jean-Pierre; Gessain, Antoine; Schwartz, Olivier; Ozden, Simona

    2006-01-01

    Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism. PMID:16641270

  20. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    SciTech Connect

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  1. Increasing the physical size and nucleation status of human pluripotent stem cell-derived ventricular cardiomyocytes by cell fusion.

    PubMed

    Kong, Chi-Wing; Chen, Shuxun; Geng, Lin; Shum, Angie Man-Yee; Sun, Dong; Li, Ronald A

    2017-03-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide an unlimited source of donor cells for potential cardiac regenerative therapies. However, hPSC-CMs are immature. For instance, hPSC-CMs are only 1/10 of the physical size of their adult counterparts; the majority are mono- rather than bi- or multi-nucleated, which is an evolutionary adaptive feature in metabolically active cells such as adult CMs. Here, we attempted to increase the physical size and nucleation status of hPSC-derived ventricular (V) cardiomyocytes (hPSC-VCMs) using chemically-induced cell fusion, and examined the subsequent functional effects. Polyethylene glycol (PEG) was employed to fuse a 1:1 mixture of lentiviral vectors LV-MLC2v-GFP- or -tdTomato-labeled hPSC-VCMs, such that hPSC-VCMs fused syncytia (FS) were identified as doubly GFP(+)/tdTomato(+) multi-nucleated cells. These microscopically-identified FS were doubled in size as gauged by their capacitance when compared to the control mononucleated hPSC-VCMs using patch-clamp analysis. Reduced automaticity or action potential (AP) firing rate and moderately prolonged AP duration were observed in FS from day 6 post-fusion induction. However, Ca(2+) handling, mitochondrial biogenesis and the extent of apoptosis were not significantly altered. We conclude that larger, multi-nucleated hPSC-VCMs FS can be created by chemically-induced cell fusion but global maturation requires additional triggering cues.

  2. Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes

    PubMed Central

    1978-01-01

    Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'- tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++- independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259). PMID:211140

  3. Laser-induced tissue fluorescence in radiofrequency tissue-fusion characterization

    NASA Astrophysics Data System (ADS)

    Su, Lei; Fonseca, Martina B.; Arya, Shobhit; Kudo, Hiromi; Goldin, Robert; Hanna, George B.; Elson, Daniel S.

    2014-01-01

    Heat-induced tissue fusion is an important procedure in modern surgery and can greatly reduce trauma, complications, and mortality during minimally invasive surgical blood vessel anastomosis, but it may also have further benefits if applied to other tissue types such as small and large intestine anastomoses. We present a tissue-fusion characterization technology using laser-induced fluorescence spectroscopy, which provides further insight into tissue constituent variations at the molecular level. In particular, an increase of fluorescence intensity in 450- to 550-nm range for 375- and 405-nm excitation suggests that the collagen cross-linking in fused tissues increased. Our experimental and statistical analyses showed that, by using fluorescence spectral data, good fusion could be differentiated from other cases with an accuracy of more than 95%. This suggests that the fluorescence spectroscopy could be potentially used as a feedback control method in online tissue-fusion monitoring.

  4. The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation.

    PubMed

    Key, Tim; Sarker, Muzaddid; de Antueno, Roberto; Rainey, Jan K; Duncan, Roy

    2015-02-01

    The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell-cell, rather than virus-cell, membrane fusion. The 36-40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell-cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome-liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

  5. Chronic sun exposure-related fusion oncogenes EGFR-PPARGC1A in cutaneous squamous cell carcinoma.

    PubMed

    Egashira, Sho; Jinnin, Masatoshi; Ajino, Manami; Shimozono, Naoki; Okamoto, Sayo; Tasaki, Yukino; Hirano, Ayaka; Ide, Maho; Kajihara, Ikko; Aoi, Jun; Harada, Miho; Igata, Toshikatsu; Masuguchi, Shinichi; Fukushima, Satoshi; Ihn, Hironobu

    2017-10-04

    Cutaneous squamous cell carcinoma (cSCC) differs from SCC of other organs in its strong association with chronic sun exposure. However, the specific driver mutations in cSCC remain unknown. Fusion genes in established cSCC cell lines (A431 and DJM-1) were predicted by transcriptome sequence, and validated by Sanger sequence, fluorescence in situ hybridization and G-banding. By transcriptome sequencing, we identified fusion gene EGFR-PPARGC1A in A431, which were expressed in 31 of 102 cSCCs. The lesions harboring the fusion gene tended to be located in sun-exposed areas. In vivo cutaneous implantation of EGFR-PPARGC1A-expressing NIH3T3 induced tumors resembling human cSCC, indicating its potent tumorigenicity. NIH3T3 transfected with EGFR-PPARGC1A as well as A431 showed increased cell proliferation activity. With regard to underlying mechanism, EGFR-PPARGC1A protein causes constitutive tyrosine phosphorylation, and induces the phosphorylation of wild-type full-length epidermal growth factor receptor (EGFR) by dimerization. Conversely, the RNAi-mediated attenuation of EGFR or CRISPR/Cas9-mediated knockdown of the fusion gene in A431 led to a decrease in the cell number, and may have therapeutic value. Our findings advance the knowledge concerning genetic causes of cSCC and the function of EGFR, with potential implications for new diagnostic and therapeutic approaches.

  6. Human trophoblast in trisomy 21: a model for cell-cell fusion dynamic investigation.

    PubMed

    Malassiné, André; Pidoux, Guillaume; Gerbaud, Pascale; Frendo, Jean Louis; Evain-Brion, Danièle

    2011-01-01

    Trophoblastic cell fusion is one essential step of the human trophoblast differentiation leading to formation of the syncytiotrophoblast, site of the numerous placental functions. This process is multifactorial and finely regulated. Using the physiological model of primary culture of trophoblastic cells isolated from human placenta, we have identified different membrane proteins directly involved in trophoblastic cell fusion: connexin 43, ZO-1 and recently syncytins. These fusogenic membrane retroviral envelop glycoproteins: syncytin-1 (encoded by the HERV-W gene) and syncytin-2 (encoded by the FRD gene) and their receptors are major factors involved in human placental development. Disturbances of syncytiotrophoblast formation are observed in trisomy 21-affected placentas. Overexpression of the copper/zinc superoxide dismutase (SOD-1), encoded by chromosome 21 as well as an abnormal hCG signaling are implicated in the defect of syncytiotrophoblast formation. This abnormal trophoblast fusion and differentiation in trisomy 21-affected placenta is reversible in vitro by different ways.

  7. Characterization of the plasma membrane localization and orientation of HPV16 E5 for cell-cell fusion

    SciTech Connect

    Hu Lulin; Ceresa, Brian P.

    2009-10-10

    Human papillomavirus (HPV) is a non-enveloped DNA virus with an approx 8000 base pair genome. Infection with certain types of HPV is associated with cervical cancer, although the molecular mechanism by which HPV induces carcinogenesis is poorly understood. Three genes encoded by HPV16 are regarded as oncogenic - E5, E6, and E7. The role of E5 has been controversial. Expression of HPV16 E5 causes cell-cell fusion, an event that can lead to increased chromosomal instability, particularly in the presence of cell cycle checkpoint inhibitors like HPV16 E6 and E7. Using biochemical and cell biological assays to better understand HPV16 E5, we find that HPV16 E5 localizes to the plasma membrane with an intracellular amino terminus and an extracellular carboxyl-terminus. Further, HPV16 E5 must be expressed on both cells for cell fusion to occur. When the extracellular epitope of HPV16 E5 is targeted with an antibody, the number of bi-nucleated cells decreases.

  8. TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate.

    PubMed

    Guo, Charles C; Dancer, Jane Y; Wang, Yan; Aparicio, Ana; Navone, Nora M; Troncoso, Patricia; Czerniak, Bogdan A

    2011-01-01

    Recent studies have shown that most prostate cancers carry the TMPRSS2-ERG gene fusion. Here we evaluated the TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate (n = 12) in comparison with small cell carcinoma of the urinary bladder (n = 12) and lung (n = 11). Fluorescence in situ hybridization demonstrated rearrangement of the ERG gene in 8 cases of prostatic small cell carcinoma (67%), and the rearrangement was associated with deletion of the 5' ERG gene in 7 cases, but rearrangement of the ERG gene was not present in any small cell carcinoma of the urinary bladder or lung. Next we evaluated the TMPRSS2-ERG gene fusion in nude mouse xenografts that were derived from 2 prostatic small cell carcinomas carrying the TMPRSS2-ERG gene fusion. Two transcripts encoded by the TMPRSS2-ERG gene fusion were detected by reverse transcriptase polymerase chain reaction, and DNA sequencing demonstrated that the 2 transcripts were composed of fusions of exon 1 of the TMPRSS2 gene to exon 4 or 5 of the ERG gene. Our study demonstrates the specific presence of TMPRSS2-ERG gene fusion in prostatic small cell carcinoma, which may be helpful in distinguishing small cell carcinoma of prostatic origin from nonprostatic origins. The high prevalence of the TMPRSS2-ERG gene fusion in prostatic small cell carcinoma as well as adenocarcinoma implies that small cell carcinoma may share a common pathogenic pathway with adenocarcinoma in the prostate.

  9. Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells.

    PubMed

    Ishihara, Naotada; Jofuku, Akihiro; Eura, Yuka; Mihara, Katsuyoshi

    2003-02-21

    Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.

  10. Cell fusion studies to examine the mechanism for etoposide resistance in Chinese hamster V79 spheroids.

    PubMed

    Luo, C; Johnston, P J; MacPhail, S H; Banáth, J P; Oloumi, A; Olive, P L

    1998-09-15

    When exposed to etoposide, the outer cells from Chinese hamster V79 spheroids are about 10 times more resistant to DNA strand breaks and cell killing than V79 cells grown as monolayers. Previous results have shown that the outer cells of both spheroids and monolayers grow at the same rate and contain the same amount and activity of the target enzyme, topoisomerase II. In order to examine possible mechanisms for this resistance, cell fusion studies were conducted with fluorescent dye-tagged monolayer and spheroid cells. Fused cells were exposed for 30 min to 1.2 microg/ml etoposide and then separated using fluorescence-activated cell sorting into binucleate cells consisting of two monolayer cells, two spheroid cells, or a mixed doublet consisting of one cell of each type. Individual sorted cell doublets were examined for the presence of etoposide-induced DNA strand breaks using the alkaline comet assay. As expected, doublets of monolayer cells were sensitive to etoposide and doublets of spheroid cells were resistant. However, mixed doublets were as resistant to DNA damage by etoposide as spheroid doublets. In comparison, when etoposide- or adriamycin-resistant V79 monolayer cells were fused to the parent monolayer cells, the expected intermediate sensitivity to etoposide was observed for the mixed doublets. We conclude that etoposide resistance associated with the outer cells of spheroids can be "transferred" to produce resistance in monolayer cells. Rapid changes in phosphorylation that can affect topoisomerase II activity or localization, or that can alter chromatin structure, are suggested as possible mechanisms of resistance. In support of this hypothesis, topo IIalpha phosphorylation was at least 10 times greater in monolayers than in the outer cell layer of spheroids. Copyright 1998 Academic Press.

  11. Dual-specificity tyrosine phosphorylation-regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner.

    PubMed

    Guterman-Ram, Gali; Pesic, Milena; Orenbuch, Ayelet; Czeiger, Tal; Aflalo, Anastasia; Levaot, Noam

    2018-01-01

    Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells. © 2017 Wiley Periodicals, Inc.

  12. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer.

    PubMed

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga; Savova, Virginia; Krzystanek, Marcin; Li, Lewyn; Marusyk, Andriy; Tabassum, Doris P; Zak, Alexander; Flacker, Mary Jo; Li, Mei; Lin, Jessica J; Sukumar, Saraswati; Suzuki, Hiromu; Long, Henry; Szallasi, Zoltan; Gimelbrant, Alexander; Maruyama, Reo; Polyak, Kornelia

    2015-06-16

    Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

  13. Dual Wavelength Imaging Allows Analysis of Membrane Fusion of Influenza Virus inside Cells

    PubMed Central

    Sakai, Tatsuya; Ohuchi, Masanobu; Imai, Masaki; Mizuno, Takafumi; Kawasaki, Kazunori; Kuroda, Kazumichi; Yamashina, Shohei

    2006-01-01

    Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion. PMID:16439557

  14. Intermediates in influenza virus PR/8 haemagglutinin-induced membrane fusion.

    PubMed

    Pak, C C; Krumbiegel, M; Blumenthal, R

    1994-02-01

    The fusion kinetics with erythrocyte ghosts of two influenza A virus strains, A/Aichi/2/68 (X:31) and A/PR/8/34 (PR/8), were compared and correlated with the kinetics of haemagglutinin (HA) conformational change. Previously it had been shown that X:31 fuses with liposomes or erythrocytes at 4 degrees C, pH 5 after a lag time of 5 to 10 min whereas PR/8 displayed no fusion with liposomes at that temperature. We have confirmed the absence of cold fusion by PR/8 with erythrocyte ghosts. In contrast to X:31, PR/8 could not be committed to fuse at neutral pH and 37 degrees C by a preincubation at low pH and 4 degrees C. To examine whether the lack of commitment and cold fusion were due to a failure of PR/8 HA to undergo conformational changes at low temperature and pH, we analysed susceptibility of HA to proteinase K digestion, liposome binding to the virus, and immunoprecipitations of HA with conformation-specific antibodies. Although there was little binding of PR/8 to liposomes at 4 degrees C and pH 5, we did observe exposure of the fusion peptide. This study reveals a low temperature intermediate in membrane fusion exhibited by the HA of influenza virus strain PR/8, which involves low pH-induced conformational changes including exposure of the fusion peptide with little interaction of HA with the target membrane.

  15. Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

    SciTech Connect

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    2009-10-09

    Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26{sup tm1Sor}, revealed that treatment of 1-cell or 2-cell embryos with 3 {mu}M of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

  16. R468A mutation in perfringolysin O destabilizes toxin structure and induces membrane fusion.

    PubMed

    Kulma, Magdalena; Kacprzyk-Stokowiec, Aleksandra; Kwiatkowska, Katarzyna; Traczyk, Gabriela; Sobota, Andrzej; Dadlez, Michał

    2017-06-01

    Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. Upon binding to a cholesterol-containing membrane, PFO undergoes a series of structural changes that result in the formation of a β-barrel pore and cell lysis. Recognition and binding to cholesterol are mediated by the D4 domain, one of four domains of PFO. The D4 domain contains a conserved tryptophan-rich loop named undecapeptide (E458CTGLAWEWWR468) in which arginine 468 is essential for retaining allosteric coupling between D4 and other domains during interaction of PFO with the membrane. In this report we studied the impact of R468A mutation on the whole protein structure using hydrogen-deuterium exchange coupled with mass spectrometry. We found that in aqueous solution, compared to wild type (PFO), PFO(R468A) showed increased deuterium uptake due to exposure of internal toxin regions to the solvent. This change reflected an overall structural destabilization of PFO(R468A) in solution. Conversely, upon binding to cholesterol-containing membranes, PFO(R468A) revealed a profound decrease of hydrogen-deuterium exchange when compared to PFO. This block of deuterium uptake resulted from PFO(R468A)-induced aggregation and fusion of liposomes, as found by dynamic light scattering, microscopic observations and FRET measurements. In the result of liposome aggregation and fusion, the entire PFO(R468A) molecule became shielded from aqueous solution and thereby was protected against proteolytic digestion and deuteration. We have established that structural changes induced by the R468A mutation lead to exposure of an additional cholesterol-independent liposome-binding site in PFO that confers its fusogenic property, altering the mode of the toxin action. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    PubMed

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  18. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  19. Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

    2005-05-01

    Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

  20. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

    PubMed

    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

    2016-10-18

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

  1. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    PubMed

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  2. Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

    PubMed

    Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

    2015-09-01

    The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

  3. Development of a lentivirus vector-based assay for non-destructive monitoring of cell fusion activity.

    PubMed

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J; de Vries, Antoine A F

    2014-01-01

    Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS-) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying

  4. Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

    PubMed Central

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J.; de Vries, Antoine A. F.

    2014-01-01

    Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS−) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS− both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS− while at low acceptor-to-donor cell ratios FLPeNLS− was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for

  5. Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

    SciTech Connect

    Zhang, Wenbin; Chen, Yaomin; Yang, Qun; Che, Honglei; Chen, Xiangjun; Yao, Ting; Zhao, Fang; Liu, Mingchao; Ke, Tao; Chen, Jingyuan; Luo, Wenjing

    2010-06-25

    Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 {+-} 0.1 {sup o}C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.

  6. Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells.

    PubMed

    Zhang, Wenbin; Chen, Yaomin; Yang, Qun; Che, Honglei; Chen, Xiangjun; Yao, Ting; Zhao, Fang; Liu, Mingchao; Ke, Tao; Chen, Jingyuan; Luo, Wenjing

    2010-06-25

    Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0+/-0.1 degrees C) for 2, 4, 8, or 12h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.

  7. Novel Oxysterols Have Pro-Osteogenic and Anti-Adipogenic Effects In Vitro and Induce Spinal Fusion In Vivo

    PubMed Central

    Johnson, Jared S.; Meliton, Vicente; Kim, Woo Kyun; Lee, Kwang-Bok; Wang, Jeffrey C.; Nguyen, KhanhLinh; Yoo, Dongwon; Jung, Michael E.; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C.; Magyar, Clara; Nargizyan, Taya; Hahn, Theodore J.; Farouz, Francine; Thies, Scott; Parhami, Farhad

    2011-01-01

    Stimulation of bone formation by osteoinductive materials is of great clinical importance in spinal fusion surgery, repair of bone fractures, and in the treatment of osteoporosis. We previously reported that specific naturally occurring oxysterols including 20(S)-hydroxycholesterol (20S) induce the osteogenic differentiation of pluripotent mesenchymal cells, while inhibiting their adipogenic differentiation. Here we report the characterization of two structural analogs of 20S, Oxy34 and Oxy49, which induce the osteogenic and inhibit the adipogenic differentiation of bone marrow stromal cells (MSC) through activation of Hedgehog (Hh) signaling. Treatment of M2-10B4 MSC with Oxy34 or Oxy49 induced the expression of osteogenic differentiation markers Runx2, Osterix (Osx), alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OCN), as well as ALP enzymatic activity and robust mineralization. Treatment with oxysterols together with PPARγ activator, troglitazone (Tro), inhibited mRNA expression for adipogenic genes PPARγ, LPL, and aP2, and inhibited the formation of adipocytes. Efficacy of Oxy34 and Oxy49 in stimulating bone formation in vivo was assessed using the posterolateral intertransverse process rat spinal fusion model. Rats receiving collagen implants with Oxy 34 or Oxy49 showed comparable osteogenic efficacy to BMP2/collagen implants as measured by radiography, MicroCT, and manual inspection. Histological analysis showed trabecular and cortical bone formation by oxysterols and rhBMP2 within the fusion mass, with robust adipogenesis in BMP2-induced bone and significantly less adipocytes in oxysterol-induced bone. These data suggest that Oxy34 and Oxy49 are effective novel osteoinductive molecules and may be suitable candidates for further development and use in orthopaedic indications requiring local bone formation. PMID:21503957

  8. Tracking fusion of human mesenchymal stem cells after transplantation to the heart.

    PubMed

    Freeman, Brian T; Kouris, Nicholas A; Ogle, Brenda M

    2015-06-01

    Evidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease. Mesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i

  9. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  10. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  11. Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review).

    PubMed

    Sinkovics, Joseph G

    2009-09-01

    Evolving young genomes of archaea, prokaryota and unicellular eukaryota were wide open for the acceptance of alien genomic sequences, which they often preserved and vertically transferred to their descendants throughout three billion years of evolution. Established complex large genomes, although seeded with ancestral retroelements, have come to regulate strictly their integrity. However, intruding retroelements, especially the descendents of Ty3/Gypsy, the chromoviruses, continue to find their ways into even the most established genomes. The simian and hominoid-Homo genomes preserved and accommodated a large number of endogenous retroviral genomic segments. These retroelements may mature into exogenous retroviruses, or into functional new genes. Phages and viruses have been instrumental in incorporating and transferring host cell genes. These events profoundly influenced and altered the course of evolution. Horizontal (lateral) gene transfers (HGT) overwhelmed the genomes of the ancient protocells and the evolving unicellular microorganisms, actually leading to their Cambrian explosion. While the rigidly organized genomes of multicellular organisms increasingly resist H/LGT, de-differentiated cells assuming the metabolism of their onto- or phylogenetic ancestors, open up widely to the practice of H/LGT by direct transfer, or to transfers mediated by viruses, or by cell fusions. This activity is intensified in malignantly transformed cells, thus rendering these subjects receptive to therapy with oncolytic viruses and with viral vectors of tumor-suppressive or immunogenic genetic materials. Naturally formed hybrids of dendritic and tumor cells are often tolerogenic, whereas laboratory products of these unisons may be immunogenic in the hosts of origin. As human breast cancer stem cells are induced by a treacherous class of CD8+ T cells to undergo epithelial to mesenchymal (ETM) transition and to yield to malignant transformation by the omnipresent proto

  12. A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

    SciTech Connect

    Rowe, Cynthia L.; Connolly, Sarah A.; Chen, Jia; Jardetzky, Theodore S.; Longnecker, Richard

    2013-02-05

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

  13. Expression of the A56 and K2 proteins is sufficient to inhibit vaccinia virus entry and cell fusion.

    PubMed

    Wagenaar, Timothy R; Moss, Bernard

    2009-02-01

    Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

  14. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

    PubMed

    Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

    2015-10-06

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

  15. Novel TRAF1-ALK fusion identified by deep RNA sequencing of anaplastic large cell lymphoma.

    PubMed

    Feldman, Andrew L; Vasmatzis, George; Asmann, Yan W; Davila, Jaime; Middha, Sumit; Eckloff, Bruce W; Johnson, Sarah H; Porcher, Julie C; Ansell, Stephen M; Caride, Ariel

    2013-11-01

    Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high-throughput, "deep" sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1-ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50-year-old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non-NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1-ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1-ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc.

  16. Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

    PubMed

    Simic, Damir; Euler, Catherine; Thurby, Christina; Peden, Mike; Tannehill-Gregg, Sarah; Bunch, Todd; Sanderson, Thomas; Van Vleet, Terry

    2012-08-01

    In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion. © 2012 by John Wiley & Sons, Inc.

  17. Identification of kinase fusion oncogenes in post-Chernobyl radiation-induced thyroid cancers.

    PubMed

    Ricarte-Filho, Julio C; Li, Sheng; Garcia-Rendueles, Maria E R; Montero-Conde, Cristina; Voza, Francesca; Knauf, Jeffrey A; Heguy, Adriana; Viale, Agnes; Bogdanova, Tetyana; Thomas, Geraldine A; Mason, Christopher E; Fagin, James A

    2013-11-01

    Exposure to ionizing radiation during childhood markedly increases the risk of developing papillary thyroid cancer. We examined tissues from 26 Ukrainian patients with thyroid cancer who were younger than 10 years of age and living in contaminated areas during the time of the Chernobyl nuclear reactor accident. We identified nonoverlapping somatic driver mutations in all 26 cases through candidate gene assays and next-generation RNA sequencing. We found that 22 tumors harbored fusion oncogenes that arose primarily through intrachromosomal rearrangements. Altogether, 23 of the oncogenic drivers identified in this cohort aberrantly activate MAPK signaling, including the 2 somatic rearrangements resulting in fusion of transcription factor ETS variant 6 (ETV6) with neurotrophic tyrosine kinase receptor, type 3 (NTRK3) and fusion of acylglycerol kinase (AGK) with BRAF. Two other tumors harbored distinct fusions leading to overexpression of the nuclear receptor PPARγ. Fusion oncogenes were less prevalent in tumors from a cohort of children with pediatric thyroid cancers that had not been exposed to radiation but were from the same geographical regions. Radiation-induced thyroid cancers provide a paradigm of tumorigenesis driven by fusion oncogenes that activate MAPK signaling or, less frequently, a PPARγ-driven transcriptional program.

  18. Identification of kinase fusion oncogenes in post-Chernobyl radiation-induced thyroid cancers

    PubMed Central

    Ricarte-Filho, Julio C.; Li, Sheng; Garcia-Rendueles, Maria E.R.; Montero-Conde, Cristina; Voza, Francesca; Knauf, Jeffrey A.; Heguy, Adriana; Viale, Agnes; Bogdanova, Tetyana; Thomas, Geraldine A.; Mason, Christopher E.; Fagin, James A.

    2013-01-01

    Exposure to ionizing radiation during childhood markedly increases the risk of developing papillary thyroid cancer. We examined tissues from 26 Ukrainian patients with thyroid cancer who were younger than 10 years of age and living in contaminated areas during the time of the Chernobyl nuclear reactor accident. We identified nonoverlapping somatic driver mutations in all 26 cases through candidate gene assays and next-generation RNA sequencing. We found that 22 tumors harbored fusion oncogenes that arose primarily through intrachromosomal rearrangements. Altogether, 23 of the oncogenic drivers identified in this cohort aberrantly activate MAPK signaling, including the 2 somatic rearrangements resulting in fusion of transcription factor ETS variant 6 (ETV6) with neurotrophic tyrosine kinase receptor, type 3 (NTRK3) and fusion of acylglycerol kinase (AGK) with BRAF. Two other tumors harbored distinct fusions leading to overexpression of the nuclear receptor PPARγ. Fusion oncogenes were less prevalent in tumors from a cohort of children with pediatric thyroid cancers that had not been exposed to radiation but were from the same geographical regions. Radiation-induced thyroid cancers provide a paradigm of tumorigenesis driven by fusion oncogenes that activate MAPK signaling or, less frequently, a PPARγ-driven transcriptional program. PMID:24135138

  19. Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle

    SciTech Connect

    Gao, Kai; Zhang, Yong; Lou, Jizhong

    2016-05-13

    Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminal and C-terminal helix. -- Highlights: •Unfolding of HIV-1 gp41 six-helix bundle is studied by molecular dynamics simulations. •Specific interactions responsible for the stability of HIV-1 envelope post-fusion conformation were identified. •The gp41 six-helix bundle transition inducing membrane fusion might be a cooperative process of the three subunits.

  20. Cell fusion between gastric epithelial cells and mesenchymal stem cells results in epithelial-to-mesenchymal transition and malignant transformation.

    PubMed

    He, Xianghui; Li, Baosong; Shao, Yang; Zhao, Na; Hsu, Yiling; Zhang, Zhixiang; Zhu, Liwei

    2015-01-30

    The discovery of cancer stem cells and tumor heterogeneity prompted the exploration of additional mechanisms aside from genetic mutations for carcinogenesis and cancer progression. The aim of the present study was to investigate the effect of cell fusion between mesenchymal stem cells and the gastric epithelial cells in tumorigenesis. Cell fusion between cord blood mesenchymal stem cells and human gastric epithelial cells was performed in vitro. Cell scratch and transwell assays were performed to determine migration and invasion abilities of the hybrids. The expressions of epithelial-mesenchymal transition-related proteins and genes were analyzed by immunocytochemistry and real time quantitative PCR. Tumorigenesis of the hybrids was evaluated through in vivo inoculation in nude mice. Hybrids expressed the phenotypes of both donor cells. Aneuploidy was observed in 84.1% of cells. The hybrids showed increased proliferation, migration and invasion abilities compared with the parental cells. In addition, the expression of N-cadherin and vimentin in the hybrids was significantly higher than that of the epithelial cells, and the mRNA expression of the epithelial-mesenchymal transition-related genes, Twist and Slug, in the hybrids was also increased compared with that of the parental epithelial cells. Furthermore, the hybrids formed masses of epithelial origin with glandular structures in BALB/c nude mice. These findings suggest that cell fusion between gastric epithelial cells and mesenchymal stem cells may result in epithelial to mesenchymal transition and malignant transformation.

  1. The Influenza Hemagglutinin Fusion Domain Is an Amphipathic Helical Hairpin That Functions by Inducing Membrane Curvature*

    PubMed Central

    Smrt, Sean T.; Draney, Adrian W.; Lorieau, Justin L.

    2015-01-01

    The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk. PMID:25398882

  2. Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

    PubMed

    Ding, Xiaohui; Zhang, Xiujuan; Chong, Huihui; Zhu, Yuanmei; Wei, Huamian; Wu, Xiyuan; He, Jinsheng; Wang, Xinquan; He, Yuxian

    2017-09-15

    The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor

  3. Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.

    PubMed

    Karbowski, Mariusz; Cleland, Megan M; Roelofs, Brian A

    2014-01-01

    Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis, and stress-induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assays rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

  4. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    PubMed

    Liao, Jen-Chung

    2016-07-05

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits.

  5. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  6. Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

    PubMed

    Huerta, Leonor; López-Balderas, Nayali; Larralde, Carlos; Lamoyi, Edmundo

    2006-12-01

    Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

  7. Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

    PubMed

    Powell, Anne E; Anderson, Eric C; Davies, Paige S; Silk, Alain D; Pelz, Carl; Impey, Soren; Wong, Melissa H

    2011-02-15

    The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells. ©2011 AACR.

  8. Spine fusion using cell matrix composites enriched in bone marrow-derived cells.

    PubMed

    Muschler, George F; Nitto, Hironori; Matsukura, Yoichi; Boehm, Cynthia; Valdevit, Antonio; Kambic, Helen; Davros, William; Powell, Kimerly; Easley, Kirk

    2003-02-01

    Bone marrow-derived cells including osteoblastic progenitors can be concentrated rapidly from bone marrow aspirates using the surface of selected implantable matrices for selective cell attachment. Concentration of cells in this way to produce an enriched cellular composite graft improves graft efficacy. The current study was designed to test the hypothesis that the biologic milieu of a bone marrow clot will significantly improve the efficacy of such a graft. An established posterior spinal fusion model and cancellous bone matrix was used to compare an enriched cellular composite bone graft alone, bone matrix plus bone marrow clot, and an enriched bone matrix composite graft plus bone marrow clot. Union score, quantitative computed tomography, and mechanical testing were used to define outcome. The union score for the enriched bone matrix plus bone marrow clot composite was superior to the enriched bone matrix alone and the bone matrix plus bone marrow clot. The enriched bone matrix plus bone marrow clot composite also was superior to the enriched bone matrix alone in fusion volume and in fusion area. These data confirm that the addition of a bone marrow clot to an enriched cell-matrix composite graft results in significant improvement in graft performance. Enriched composite grafts prepared using this strategy provide a rapid, simple, safe, and inexpensive method for intraoperative concentration and delivery of bone marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting.

  9. Polyethylene glycol-mediated fusion of herpes simplex type 1 virions with the plasma membrane of cells that support endocytic entry.

    PubMed

    Walker, Erik B; Pritchard, Suzanne M; Cunha, Cristina W; Aguilar, Hector C; Nicola, Anthony V

    2015-11-16

    Mouse B78 cells and Chinese hamster ovary (CHO) cells are important to the study of HSV-1 entry because both are resistant to infection at the level of viral entry. When provided with a gD-receptor such as nectin-1, these cells support HSV-1 entry by an endocytosis pathway. Treating some viruses bound to cells with the fusogen polyethylene glycol (PEG) mediates viral fusion with the cell surface but is insufficient to rescue viral entry. It is unclear whether PEG-mediated fusion of HSV with the plasma membrane of B78 or CHO cells results in successful entry and infection. Treating HSV-1 bound to B78 or CHO cells with PEG allowed viral entry as measured by virus-induced beta-galactosidase activity. Based on the mechanism of PEG action, we propose that entry likely proceeds by direct fusion of HSV particles with the plasma membrane. Under the conditions tested, PEG-mediated infection of CHO cells progressed to the level of HSV late gene expression, while B78 cells supported HSV DNA replication. We tested whether proteolysis or acidification of cell-bound virions could trigger HSV fusion with the plasma membrane. Under the conditions tested, mildly acidic pH of 5-6 or the protease trypsin were not capable of triggering HSV-1 fusion as compared to PEG-treated cell-bound virions. B78 cells and CHO cells, which typically endocytose HSV prior to viral penetration, are capable of supporting HSV-1 entry via direct penetration. HSV capsids delivered directly to the cytosol at the periphery of these cells complete the entry process. B78 and CHO cells may be utilized to screen for factors that trigger entry as a consequence of fusion of virions with the cell surface, and PEG treatment can provide a necessary control.

  10. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  11. The temperature arrested intermediate of virus-cell fusion is a functional step in HIV infection.

    PubMed

    Henderson, Hamani I; Hope, Thomas J

    2006-05-25

    HIV entry occurs via membrane-mediated fusion of virus and target cells. Interactions between gp120 and cellular co-receptors lead to both the formation of fusion pores and release of the HIV genome into target cells. Studies using cell-cell fusion assays have demonstrated that a temperature-arrested state (TAS) can generate a stable intermediate in fusion related events. Other studies with MLV pseudotyped with HIV envelope also found that a temperature sensitive intermediate could be generated as revealed by the loss of a fluorescently labeled membrane. However, such an intermediate has never been analyzed in the context of virus infection. Therefore, we used virus-cell infection with replication competent HIV to gain insights into virus-cell fusion. We find that the TAS is an intermediate in the process culminating in the HIV infection of a target cell. In the virion-cell TAS, CD4 has been engaged, the heptad repeats of gp41 are exposed and the complex is kinetically predisposed to interact with coreceptor to complete the fusion event leading to infection.

  12. Fusion reactions in collisions induced by Li isotopes on Sn targets

    SciTech Connect

    Fisichella, M.; Shotter, A. C.; Di Pietro, A.; Figuera, P.; Lattuada, M.; Marchetta, C.; Musumarra, A.; Pellegriti, M. G.; Ruiz, C.; Scuderi, V.; Strano, E.; Torresi, D.; Zadro, M.

    2012-10-20

    Fusion cross sections for the {sup 6}Li+{sup 120}Sn and {sup 7}Li+{sup 119}Sn systems have been measured. We aim to search for possible effects due to the different neutron transfer Q-values, by comparing the fusion cross sections for the two systems below the barrier. This experiment is the first step of a wider systematic aiming to study the above problems in collisions induced by stable and unstable Li isotopes on tin all forming the same compound nucleus.

  13. Possibility of phase transitions inducing cold fusion in palladium/deuterium systems

    SciTech Connect

    Zhang, W.X. )

    1992-01-01

    In this paper a tentative theory is presented in which {beta}-phase PdD{sub x} containing supersaturated deuterium transits into {beta}-phase PdD{sub x} containing less deuterium and {alpha}-phase PdD{sub x}. High-pressure ({approx}10 GPa) deuterium bubbles form at the same time. As the bubbles release energy, cracks are created in the PdD{sub x} crystal, and charge separation of deuterium occurs. Thus would cold fusion be induced. This proposal supports the fracture mechanism for cold fusion.

  14. Viral and developmental cell fusion mechanisms: conservation and divergence.

    PubMed

    Sapir, Amir; Avinoam, Ori; Podbilewicz, Benjamin; Chernomordik, Leonid V

    2008-01-01

    Membrane fusion is a fundamental requirement in numerous developmental, physiological, and pathological processes in eukaryotes. So far, only a limited number of viral and cellular fusogens, proteins that fuse membranes, have been isolated and characterized. Despite the diversity in structures and functions of known fusogens, some common principles of action apply to all fusion reactions. These can serve as guidelines in the search for new fusogens, and may allow the formulation of a cross-species, unified theory to explain divergent and convergent evolutionary principles of membrane fusion.

  15. Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

    PubMed

    Leemans, A; De Schryver, M; Van der Gucht, W; Heykers, A; Pintelon, I; Hotard, A L; Moore, M L; Melero, J A; McLellan, J S; Graham, B S; Broadbent, L; Power, U F; Caljon, G; Cos, P; Maes, L; Delputte, P

    2017-07-15

    Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are

  16. Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

    PubMed

    Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

    2016-08-01

    Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  17. Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

    PubMed Central

    Cantone, Irene; Bagci, Hakan; Dormann, Dirk; Dharmalingam, Gopuraja; Nesterova, Tatyana; Brockdorff, Neil; Rougeulle, Claire; Vallot, Celine; Heard, Edith; Chaligne, Ronan; Merkenschlager, Matthias; Fisher, Amanda G.

    2016-01-01

    Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. PMID:27507283

  18. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    SciTech Connect

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  19. United we stand: Adhesion and molecular mechanisms driving cell fusion across species.

    PubMed

    Zito, Francesca; Lampiasi, Nadia; Kireev, Igor; Russo, Roberta

    2016-12-01

    Cell-cell fusion is a physiological process playing an essential role for fertilization, shaping organs, tissue repair and immune defense in multicellular organisms. Recent research in the field aims to understand why two or more cells fuse each other and to decipher the general mechanisms regulating this process. Few basic and general steps can be identified, i.e. migration, adhesion and fusion, which are common to different types of cells. As pre-fused and fused cells undergo dramatic changes in their ultrastructure and behavior, the coordinated action of multiple factors is required, including adhesion molecules, cell surface receptors, intracellular kinases, transcription factors, and miRNAs. Although a number of reviews on cell-cell fusion have been published over the years, comprehensive reviews that broadly summarize this process including extracellular and intracellular cues are lacking. For example, a link between cell fusion and adhesive molecules and/or miRNAs has rarely been highlighted in the recent literature. In this review, we will summarize some molecular mechanisms controlling the process of somatic cell-cell fusion during embryonic development. We will specially focus on adhesive molecules, ECM components and miRNAs, providing a summary of important findings on their role in mediating this process in few model systems, in vertebrate and invertebrate organisms.

  20. Evidence for cell fusion is absent in vascular lesions associated with pulmonary arterial hypertension

    PubMed Central

    Majka, S. M.; Skokan, M.; Wheeler, L.; Harral, J.; Gladson, S.; Burnham, E.; Loyd, J. E.; Stenmark, K. R.; Varella-Garcia, M.; West, J.

    2008-01-01

    Pulmonary arterial hypertension (PAH) is a fatal disease associated with severe remodeling of the large and small pulmonary arteries. Increased accumulation of inflammatory cells and apoptosis-resistant cells are contributing factors. Proliferative apoptosis-resistant cells expressing CD133 are increased in the circulation of PAH patients. Circulating cells can contribute to tissue repair via cell fusion and heterokaryon formation. We therefore hypothesized that in the presence of increased leukocytes and CD133-positive (CD133pos) cells in PAH lung tissue, cell fusion and resulting genomic instability could account for abnormal cell proliferation and the genesis of vascular lesions. We performed analyses of CD45/CD133 localization, cell fusion, and proliferation during late-stage PAH in human lung tissue from control subjects and subjects with idiopathic (IPAH) and familial (FPAH) PAH. Localization, proliferation, and quantitation of cell populations in individual patients were performed by immunolocalization. The occurrence of cellular fusion in vascular lesions was analyzed in lung tissue by fluorescence in situ hybridization. We found the accumulation of CD45pos leukocytic cells in the tissue parenchyma and perivascular regions in PAH patients and less frequently observed myeloid cells (CD45/CD11b). CD133pos cells were detected in occlusive lesions and perivascular areas in those with PAH and were more numerous in those with IPAH lesions than in FPAH lesions. Cells coexpressing CD133 and smooth muscle α-actin were occasionally observed in occlusive lesions and perivascular areas. Proliferating cells were more prominent in IPAH lesions and colocalized with CD45 or CD133. We found no evidence of increased ploidy to suggest cell fusion. Taken together, these data suggest that abnormal lesion formation in PAH occurs in the absence of cell fusion. PMID:18931051

  1. Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

    SciTech Connect

    Song, Kai; Zhu, Fei; Zhang, Han-zhong; Shang, Zheng-jun

    2012-08-15

    Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

  2. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores.

    PubMed

    Monel, Blandine; Beaumont, Elodie; Vendrame, Daniela; Schwartz, Olivier; Brand, Denys; Mammano, Fabrizio

    2012-04-01

    Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.

  3. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  4. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

    PubMed

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2014-07-10

    Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

  5. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells

    PubMed Central

    2014-01-01

    Background Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Methods Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. Results In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. Conclusions In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the

  6. FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

    SciTech Connect

    Yang, Liu; Hu, Hsien-Ming; Zielinska-Kwiatkowska, Anna; Chansky, Howard A.

    2010-11-05

    Research highlights: {yields} Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. {yields} The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. {yields} While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. {yields} This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. -- Abstract: Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

  7. Recent progress on MHD-induced loss of D-D fusion products in TFTR

    SciTech Connect

    Zweben, S.J.; Darrow, D.S.; Budny, R.V.; Cheng, C.Z.; Fredrickson, E.D.; Herrmann, H.; Mynick, H.E.; Schivell, J.; Chang, Z.

    1993-08-01

    This paper reviews the recent progress made toward understanding the MHD-induced loss of D-D fusion products which has been seen on TFTR since 1988. These measurements have been made using the ``lost alpha`` diagnostic, which is described briefly. The largest MHD- induced loss occurs with coherent 3/2 or 2/1 MHD activity (kink/tearing modes), which can cause up to {approx}3--5 times the first-orbit loss at I{approx}1.6--1.8 MA, roughly a {approx}20--30% global los of D-D fusion products. Modeling of these MHD-induced losses has progressed to the point where the basic loss mechanism can be accounted for qualitatively, but the experimental results can not yet be understood quantitatively. Several alpha loss codes are being developed to improve the quantitative comparison between experiment and theory.

  8. Modulation of non steroidal anti-inflammatory drug induced membrane fusion by copper coordination of these drugs: anchoring effect.

    PubMed

    Majumdar, Anupa; Chakraborty, Sreeja; Sarkar, Munna

    2014-12-04

    Membrane fusion, an integral event in several biological processes, is characterized by several intermediate steps guided by specific energy barriers. Hence, it requires the aid of fusogens to complete the process. Common fusogens, such as proteins/peptides, have the ability to overcome theses barriers by their conformational reorganization, an advantage not shared by small drug molecules. Hence, drug induced fusion at physiologically relevant drug concentrations is rare and occurs only in the case of the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs). To use drugs to induce and control membrane fusion in various biochemical processes requires the understanding of how different parameters modulate fusion. Also, fusion efficacy needs to be enhanced. Here we have synthesized and used Cu(II) complexes of fusogenic oxicam NSAIDs, Meloxicam and Piroxicam, to induce fusion in model membranes monitored by using DSC, TEM, steady-state, and time-resolved spectroscopy. The ability of the complexes to anchor apposing model membranes to initiate/facilitate fusion has been demonstrated. This results in better fusion efficacy compared to the bare drugs. These complexes can take the fusion to its final step. Unlike other designed membrane anchors, the role of molecular recognition and strength of interaction between molecular partners is obliterated for these preformed Cu(II)-NSAIDs.

  9. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    NASA Astrophysics Data System (ADS)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  10. The compound DATEM inhibits respiratory syncytial virus fusion activity with epithelial cells.

    PubMed

    Ohki, Shinpei; Liu, Jin-Zhou; Schaller, Joseph; Welliver, Robert C

    2003-04-01

    The effect of diacetyltartaric acid esters of mono and diglycerides (DATEM) on fusion of respiratory syncytial virus (RSV) with HEp-2 cells was studied using the R18 fluorescence dequenching fusion assay. At DATEM concentrations less than 2.0 microg/ml, the inhibition of fusion increased with the concentration of DATEM. At 2 microg/ml of DATEM, the fusion was suppressed by 80-90%. Studies examining possible mechanism of fusion-inhibition indicated that DATEM was likely adsorbed onto lipid membranes of both viral envelope and target cell membranes. Quantitative measurements of DATEM adsorption onto membranes were also performed using lipid monolayers and vesicles. The surface pressure of lipid monolayer formed at the air/aqueous interface increased as the concentration of DATEM in the monolayer subphase increased, suggesting that DATEM was inserted into the monolayer. As the concentration of DATEM in vesicle suspensions increased, electrophoretic mobility of initially uncharged lipid vesicles also increased, reflective of increased negative charge at vesicle surfaces. These results strongly suggest that the insertion of DATEM onto membranes inhibited viral fusion. DATEM may prove to be effective in limiting the infectivity of RSV by interference with the fusion of the viral envelope with target cell membranes.

  11. Use of RecA fusion proteins to induce genomic modifications in zebrafish

    PubMed Central

    Liao, Hsin-Kai; Essner, Jeffrey J.

    2011-01-01

    The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB. PMID:21266475

  12. Use of RecA fusion proteins to induce genomic modifications in zebrafish.

    PubMed

    Liao, Hsin-Kai; Essner, Jeffrey J

    2011-05-01

    The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA-DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

  13. Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    PubMed Central

    Wei, Yongwei; Zhang, Yu; Cai, Hui; Mirza, Anne M.; Iorio, Ronald M.; Peeples, Mark E.; Niewiesk, Stefan

    2014-01-01

    ABSTRACT Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif (329RGD331). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329RGD331 motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCE Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral

  14. Pristionchus pacificus vulva formation: polarized division, cell migration, cell fusion, and evolution of invagination.

    PubMed

    Kolotuev, Irina; Podbilewicz, Benjamin

    2004-02-15

    Tube formation is a widespread process during organogenesis. Specific cellular behaviors participate in the invagination of epithelial monolayers that form tubes. However, little is known about the evolutionary mechanisms of cell assembly into tubes during development. In Caenorhabditis elegans, the detailed step-to-step process of vulva formation has been studied in wild type and in several mutants. Here we show that cellular processes during vulva development, which involve toroidal cell formation and stacking of rings, are conserved between C. elegans and Pristionchus pacificus, two species of nematodes that diverged approximately 100 million years ago. These cellular behaviors are divided into phases of cell proliferation, short-range migration, and cell fusion that are temporally distinct in C. elegans but not in P. pacificus. Thus, we identify heterochronic changes in the cellular events of vulva development between these two species. We find that alterations in the division axes of two equivalent vulval cells from Left-Right cleavage in C. elegans to Anterior-Posterior division in P. pacificus can cause the formation of an additional eighth ring. Thus, orthogonal changes in cell division axes with alterations in the number and sequence of cell fusion events result in dramatic differences in vulval shape and in the number of rings in the species studied. Our characterization of vulva formation in P. pacificus compared to C. elegans provides an evolutionary-developmental foundation for molecular genetic analyses of organogenesis in different species within the phylum Nematoda.

  15. Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase.

    PubMed

    Kucerova, Lucia; Poturnajova, Martina; Tyciakova, Silvia; Matuskova, Miroslava

    2012-03-01

    Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.

  16. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    PubMed Central

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  17. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion.

    PubMed

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  19. Selective retention of bone marrow-derived cells to enhance spinal fusion.

    PubMed

    Muschler, George F; Matsukura, Yoichi; Nitto, Hironori; Boehm, Cynthia A; Valdevit, Antonio D; Kambic, Helen E; Davros, William J; Easley, Kirk A; Powell, Kimerly A

    2005-03-01

    Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal fusion model. Fusions were compared based on union score, fusion mass, fusion volume, and by mechanical testing. Enriched matrix grafts delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors than matrix mixed with bone marrow. The union score with enriched matrix was superior to matrix alone and matrix plus marrow. Fusion volume and fusion area also were greater with the enriched matrix. These data suggest that the strategy of selective retention provides a rapid, simple, and effective method for concentration and delivery of marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting procedures in various clinical settings.

  20. Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum.

    PubMed

    Ishikawa, Francine H; Souza, Elaine A; Read, Nick D; Roca, M Gabriela

    2010-01-01

    Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer. Copyright © 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  1. An Il12-Il2-Antibody Fusion Protein Targeting Hodgkin's Lymphoma Cells Potentiates Activation Of Nk And T Cells For An Anti-Tumor Attack

    PubMed Central

    Friedrichs, Björn; Heuser, Claudia; Guhlke, Stefan; Abken, Hinrich; Hombach, Andreas A.

    2012-01-01

    Successful immunotherapy of Hodgkin's disease is so far hampered by the striking unresponsiveness of lymphoma infiltrating immune cells. To mobilize both adoptive and innate immune cells for an anti-tumor attack we fused the pro-inflammatory cytokines IL2 and IL12 to an anti-CD30 scFv antibody in a dual cytokine fusion protein to accumulate both cytokines at the malignant CD30+ Hodgkin/Reed-Sternberg cells in the lymphoma lesion. The tumor-targeted IL12-IL2 fusion protein was superior in activating resting T cells to amplify and secrete pro-inflammatory cytokines compared to targeted IL2 or IL12 alone. NK cells were also activated by the dual cytokine protein to secrete IFN-γ and to lyse target cells. The tumor-targeted IL12-IL2, when applied by i.v. injection to immune-competent mice with established antigen-positive tumors, accumulated at the tumor site and induced tumor regression. Data demonstrate that simultaneous targeting of two cytokines in a spatial and temporal simultaneous fashion to pre-defined tissues is feasible by a dual-cytokine antibody fusion protein. In the case of IL12 and IL2, this produced superior anti-tumor efficacy implying the strategy to muster a broader immune cell response in the combat against cancer. PMID:23028547

  2. Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells

    PubMed Central

    1994-01-01

    To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc- expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect. PMID:8195295

  3. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    SciTech Connect

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-09-15

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  4. Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

    PubMed Central

    Urbé, Sylvie; Page, Lesley J.; Tooze, Sharon A.

    1998-01-01

    The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process. PMID:9864358

  5. Mitochondrial fusion and fission in cell life and death.

    PubMed

    Westermann, Benedikt

    2010-12-01

    Mitochondria are dynamic organelles that constantly fuse and divide. These processes (collectively termed mitochondrial dynamics) are important for mitochondrial inheritance and for the maintenance of mitochondrial functions. The core components of the evolutionarily conserved fusion and fission machineries have now been identified, and mechanistic studies have revealed the first secrets of the complex processes that govern fusion and fission of a double membrane-bound organelle. Mitochondrial dynamics was recently recognized as an important constituent of cellular quality control. Defects have detrimental consequences on bioenergetic supply and contribute to the pathogenesis of neurodegenerative diseases. These findings open exciting new directions to explore mitochondrial biology.

  6. Cold fusion, Alchemist's dream

    SciTech Connect

    Clayton, E.D.

    1989-09-01

    In this report the following topics relating to cold fusion are discussed: muon catalysed cold fusion; piezonuclear fusion; sundry explanations pertaining to cold fusion; cosmic ray muon catalysed cold fusion; vibrational mechanisms in excited states of D{sub 2} molecules; barrier penetration probabilities within the hydrogenated metal lattice/piezonuclear fusion; branching ratios of D{sub 2} fusion at low energies; fusion of deuterons into {sup 4}He; secondary D+T fusion within the hydrogenated metal lattice; {sup 3}He to {sup 4}He ratio within the metal lattice; shock induced fusion; and anomalously high isotopic ratios of {sup 3}He/{sup 4}He.

  7. Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease.

    PubMed

    Khurana, Surender; Loving, Crystal L; Manischewitz, Jody; King, Lisa R; Gauger, Phillip C; Henningson, Jamie; Vincent, Amy L; Golding, Hana

    2013-08-28

    Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

  8. Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

    PubMed

    Meertens, Nadine; Stoffel, Michael H; Cherpillod, Pascal; Wittek, Riccardo; Vandevelde, Marc; Zurbriggen, Andreas

    2003-10-01

    Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion

  9. Multi-Patterned Dynamics of Mitochondrial Fission and Fusion in a Living Cell

    PubMed Central

    Shan, Sicong; Jiang, Chunsun; Chen, Ming; Zhang, Yan; Lü, Shouqin; Chen, Juan; Zhang, Chuanmao; Chen, Quan; Long, Mian

    2012-01-01

    Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission. PMID:22649485

  10. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    PubMed

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  11. Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion.

    PubMed

    Ishihara, Naotada; Maeda, Maki; Ban, Tadato; Mihara, Katsuyoshi

    2017-04-27

    Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signaling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential, and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive, and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  12. Cluster Model for Near-barrier Fusion Induced by Weakly Bound and Halo Nuclei

    SciTech Connect

    Beck, C.; Keeley, N.

    2008-05-12

    The influence on the fusion process of coupling transfer/breakup channels is investigated for the medium weight {sup 6,7}Li+{sup 59}Co systems in the vicinity of the Coulomb barrier. Coupling effects are discussed within a comparison of predictions of the Continuum Discretized Coupled-Channels model. Applications to {sup 6}He+{sup 59}Co induced by the borromean halo nucleus {sup 6}He are also proposed.

  13. Near-barrier Fusion Induced by Stable Weakly Bound and Exotic Halo Light Nuclei

    SciTech Connect

    Beck, C.; Zafra, A. Sanchez I.; Diaz-Torres, A.; Thompson, I. J.; Keeley, N.

    2006-08-14

    The effect of breakup is investigated for the medium weight 6Li+59Co system in the vicinity of the Coulomb barrier. The strong coupling of breakup/transfer channels to fusion is discussed within a comparison of predictions of the Continuum Discretized Coupled-Channels model which is also applied to 6He+59Co a reaction induced by the borromean halo nucleus 6He.

  14. Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor α (RARα) and oncogenic RARα fusion proteins

    PubMed Central

    Zhu, Jun; Gianni, Maurizio; Kopf, Eliezer; Honoré, Nicole; Chelbi-Alix, Mounira; Koken, Marcel; Quignon, Frédérique; Rochette-Egly, Cécile; de Thé, Hugues

    1999-01-01

    Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RARα) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARα cleavage, RA triggers degradation of both PML/RARα and RARα. Similarly, in non-APL cells, RA directly targeted RARα and RARα fusions to the proteasome degradation pathway. Activation of either RARα or RXRα by specific agonists induced degradation of both proteins. Conversely, a mutation in RARα that abolishes heterodimer formation and DNA binding, blocked both RARα and RXRα degradation. Mutations in the RARα DNA-binding domain or AF-2 transcriptional activation region also impaired RARα catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation. PMID:10611294

  15. Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells.

    PubMed

    Jena, Bhanu P

    2009-07-01

    The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the 'porosome',-the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.

  16. Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

    PubMed Central

    Clawson, Gary A.; Matters, Gail L.; Xin, Ping; Imamura-Kawasawa, Yuka; Du, Zhen; Thiboutot, Diane M.; Helm, Klaus F.; Neves, Rogerio I.; Abraham, Thomas

    2015-01-01

    Background While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients. Methods We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice. Findings Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations

  17. Ankle arthrodesis fusion rates for mesenchymal stem cell bone allograft versus proximal tibia autograft.

    PubMed

    Anderson, John J; Boone, Joshua J; Hansen, Myron; Brady, Chad; Gough, Adam; Swayzee, Zflan

    2014-01-01

    Ankle arthrodesis is commonly used in the treatment of ankle arthritis. The present study compared mesenchymal stem cell (MSC) bone allografts and proximal tibia autografts as adjuncts in performing ankle arthrodesis. A total of 109 consecutive ankle fusions performed from 2002 to 2008 were evaluated retrospectively. Of the 109 fusions, 24 were excluded from the present study, leaving 85 patients who had undergone ankle arthrodesis. Of the 85 patients, 41 had received a proximal tibia autograft and 44, an MSC bone allograft. These 2 groups were reviewed and compared retrospectively at least 2 years postoperatively for the overall fusion rate, interval to radiographic fusion, and interval to clinical fusion. A modified and adjusted American College of Foot and Ankle Surgeons ankle scale was used to measure patient satisfaction. The overall fusion rate was 84.1% in the MSC bone allograft group and 95.1% in the proximal tibia autograft group (p = .158). The corresponding mean intervals to radiographic fusion were 13.0 ± 2.5 weeks and 11.3 ± 2.8 weeks (p ≤ .001). The interval to clinical fusion was 13.1 ± 2.1 weeks and 11.0 ± 1.5 weeks (p ≤ .001) in the MSC bone allograft and proximal tibia autograft group, respectively. No statistically significant difference was found in the fusion rates between the MSC bone allograft and proximal tibia autograft groups. Also, no statistically significant difference was found between the preoperative and postoperative scores using a modified and adjusted American College of Foot and Ankle Surgeons ankle scale between the 2 groups (p = .41 and p = .44, respectively). A statistically significant delay to radiographic and clinical fusion was present in the MSC bone allograft group compared with the proximal tibia autograft group; however, no difference was found in patient satisfaction.

  18. Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

    PubMed

    Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

    2016-07-08

    Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.

  19. Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

    PubMed Central

    Mito, S; Zhang, Q M; Yonei, S

    1993-01-01

    Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress. PMID:8386722

  20. Aqueous extract from a Chaga medicinal mushroom, Inonotus obliquus (higher Basidiomycetes), prevents herpes simplex virus entry through inhibition of viral-induced membrane fusion.

    PubMed

    Pan, Hong-Hui; Yu, Xiong-Tao; Li, Ting; Wu, Hong-Ling; Jiao, Chun-Wei; Cai, Mian-Hua; Li, Xiang-Min; Xie, Yi-Zhen; Wang, Yi; Peng, Tao

    2013-01-01

    Chaga medicinal mushroom, Inonotus obliquus, a popular prescription in traditional medicine in Europe and Asia, was used to reduce inflammation in the nasopharynx and to facilitate breathing. The aqueous extract from I. obliquus (AEIO) exhibited marked decrease in herpes simplex virus (HSV) infection (the 50% inhibitory concentration was 3.82 μg/mL in the plaque reduction assay and 12.29 μg/mL in the HSV-1/blue assay) as well as safety in Vero cells (the 50% cellular cytotoxicity was > 1 mg/mL, and selection index was > 80). Using a time course assay, effective stage analysis, and fusion inhibition assay, the mechanism of anti-HSV activity was found against the early stage of viral infection through inhibition of viral-induced membrane fusion. Therefore, AEIO could effectively prevent HSV-1 entry by acting on viral glycoproteins, leading to the prevention of membrane fusion, which is different from nucleoside analog antiherpetics.

  1. A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

    PubMed Central

    Hollmann, Axel; Tanner, Lukas B.; Akyol Ataman, Zeynep; Yun, Tatyana; Shui, Guanghou; Aguilar, Hector C.; Zhang, Dong; Meriwether, David; Roman-Sosa, Gleyder; Robinson, Lindsey R.; Juelich, Terry L.; Buczkowski, Hubert; Chou, Sunwen; Castanho, Miguel A. R. B.; Wolf, Mike C.; Smith, Jennifer K.; Banyard, Ashley; Kielian, Margaret; Reddy, Srinivasa; Wenk, Markus R.; Selke, Matthias; Santos, Nuno C.; Freiberg, Alexander N.; Jung, Michael E.; Lee, Benhur

    2013-01-01

    LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of 1O2 generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the

  2. Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

    SciTech Connect

    Kaleeba, Johnan A.R.; Berger, Edward A. . E-mail: edward_berger@nih.gov

    2006-10-10

    The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of {alpha}3{beta}1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor.

  3. Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

    PubMed Central

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera

    2015-01-01

    Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process. PMID:26459604

  4. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

    PubMed

    Engelholm, Lars H; Riaz, Anjum; Serra, Denise; Dagnæs-Hansen, Frederik; Johansen, Jens V; Santoni-Rugiu, Eric; Hansen, Steen H; Niola, Francesco; Frödin, Morten

    2017-09-15

    Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8 week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca

  5. The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection.

    PubMed

    Yang, Edward; Arvin, Ann M; Oliver, Stefan L

    2017-01-01

    The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (-1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been

  6. Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins.

    PubMed

    Bachran, C; Hasikova, R; Leysath, C E; Sastalla, I; Zhang, Y; Fattah, R J; Liu, S; Leppla, S H

    2014-01-16

    Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.

  7. Regulation of Fusion Pore Closure and Compound Exocytosis in Neuroendocrine PC12 Cells by SCAMP1

    PubMed Central

    Zhang, Jie; Castle, David

    2011-01-01

    During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1’s apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis. PMID:21272170

  8. HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

    SciTech Connect

    Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern; Itkin-Ansari, Pamela; Levine, Fred; E-mail: flevine@ucsd.edu

    2007-04-06

    To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.

  9. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

    PubMed

    Read, Jolene; Clancy, Eileen K; Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N; Parmar, Hiren B; Shin, Kyungsoo; Rainey, Jan K; Duncan, Roy

    2015-06-01

    Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

  10. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

    PubMed Central

    Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

    2015-01-01

    Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

  11. Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

    PubMed

    Gorokhovatsky, Andrey Yu; Marchenkov, Victor V; Rudenko, Natalia V; Ivashina, Tanya V; Ksenzenko, Vladimir N; Burkhardt, Nils; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2004-07-30

    The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

  12. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

    PubMed Central

    Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

    2016-01-01

    Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

  13. [CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer].

    PubMed

    Gong, Ting; Zhou, Hong-Li; Ba, Yi

    2013-09-01

    To investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector. CCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection. Gene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks. CCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

  14. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    PubMed

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  15. Myelin-reactive antibodies mediate the pathology of MBP-PLP fusion protein MP4-induced EAE.

    PubMed

    Kuerten, Stefanie; Pauly, Robert; Rottlaender, Andrea; Rodi, Michael; Gruppe, Traugott L; Addicks, Klaus; Tary-Lehmann, Magdalena; Lehmann, Paul V

    2011-07-01

    Experimental autoimmune encephalomyelitis (EAE) is frequently used for studies of multiple sclerosis (MS). Because in most EAE models T cells mediate the pathology in the absence of B cells/autoantibodies, the notion has evolved that also MS may be a primarily T cell-mediated disease. We have previously introduced MBP-PLP fusion protein (MP4)-induced EAE in C57BL/6 mice. Here we show that the disease in this model is antibody-dependent. Immunization of B cell-deficient mice did not induce EAE. When such B cell-deficient mice were, however, injected with MBP/PLP-specific antibodies in addition to the immunization with MP4, they developed disease of a severity and course that was similar to the wild-type mice. The deposition of antibodies in demyelinated lesions provided further evidence for the contribution of MBP/PLP-specific antibodies to CNS lesion formation. Based upon these data we suggest a two-stage model for the involvement of MBP/PLP-specific antibodies in autoimmune CNS pathology.

  16. Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

    PubMed

    Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

    2014-08-30

    Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

  17. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    SciTech Connect

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer; Bani-Yaghoub, Mahmud; Taylor, Rod; Johnston, Linda J.; Pezacki, John Paul

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  18. Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.

    PubMed

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer; Bani-Yaghoub, Mahmud; Taylor, Rod; Johnston, Linda J; Pezacki, John Paul

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize beta(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(2)AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the beta(2)AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of beta(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  19. Model-independent determination of the astrophysical S factor in laser-induced fusion plasmas

    NASA Astrophysics Data System (ADS)

    Lattuada, D.; Barbarino, M.; Bonasera, A.; Bang, W.; Quevedo, H. J.; Warren, M.; Consoli, F.; De Angelis, R.; Andreoli, P.; Kimura, S.; Dyer, G.; Bernstein, A. C.; Hagel, K.; Barbui, M.; Schmidt, K.; Gaul, E.; Donovan, M. E.; Natowitz, J. B.; Ditmire, T.

    2016-04-01

    In this work, we present a new and general method for measuring the astrophysical S factor of nuclear reactions in laser-induced plasmas and we apply it to :mmultiscripts>(d ,n )3He . The experiment was performed with the Texas Petawatt Laser, which delivered 150-270 fs pulses of energy ranging from 90 to 180 J to D2 or CD4 molecular clusters (where D denotes 2H ) . After removing the background noise, we used the measured time-of-flight data of energetic deuterium ions to obtain their energy distribution. We derive the S factor using the measured energy distribution of the ions, the measured volume of the fusion plasma, and the measured fusion yields. This method is model independent in the sense that no assumption on the state of the system is required, but it requires an accurate measurement of the ion energy distribution, especially at high energies, and of the relevant fusion yields. In the :mmultiscripts>(d ,n )3He and 3He(d ,p )4He cases discussed here, it is very important to apply the background subtraction for the energetic ions and to measure the fusion yields with high precision. While the available data on both ion distribution and fusion yields allow us to determine with good precision the S factor in the d +d case (lower Gamow energies), for the d +3He case the data are not precise enough to obtain the S factor using this method. Our results agree with other experiments within the experimental error, even though smaller values of the S factor were obtained. This might be due to the plasma environment differing from the beam target conditions in a conventional accelerator experiment.

  20. Heat generation above break-even from laser-induced fusion in ultra-dense deuterium

    SciTech Connect

    Holmlid, Leif

    2015-08-15

    Previous results from laser-induced processes in ultra-dense deuterium D(0) give conclusive evidence for ejection of neutral massive particles with energy >10 MeV u{sup −1}. Such particles can only be formed from nuclear processes like nuclear fusion at the low laser intensity used. Heat generation is of interest for future fusion energy applications and has now been measured by a small copper (Cu) cylinder surrounding the laser target. The temperature rise of the Cu cylinder is measured with an NTC resistor during around 5000 laser shots per measured point. No heating in the apparatus or the gas feed is normally used. The fusion process is suboptimal relative to previously published studies by a factor of around 10. The small neutral particles H{sub N}(0) of ultra-dense hydrogen (size of a few pm) escape with a substantial fraction of the energy. Heat loss to the D{sub 2} gas (at <1 mbar pressure) is measured and compensated for under various conditions. Heat release of a few W is observed, at up to 50% higher energy than the total laser input thus a gain of 1.5. This is uniquely high for the use of deuterium as fusion fuel. With a slightly different setup, a thermal gain of 2 is reached, thus clearly above break-even for all neutronicity values possible. Also including the large kinetic energy which is directly measured for MeV particles leaving through a small opening gives a gain of 2.3. Taking into account the lower efficiency now due to the suboptimal fusion process, previous studies indicate a gain of at least 20 during long periods.

  1. Model-independent determination of the astrophysical S factor in laser-induced fusion plasmas

    DOE PAGES

    Lattuada, D.; Barbarino, M.; Bonasera, A.; ...

    2016-04-19

    In this paper, we present a new and general method for measuring the astrophysical S factor of nuclear reactions in laser-induced plasmas and we apply it to 2H(d,n)3He. The experiment was performed with the Texas Petawatt Laser, which delivered 150–270 fs pulses of energy ranging from 90 to 180 J to D2 or CD4 molecular clusters (where D denotes 2H). After removing the background noise, we used the measured time-of-flight data of energetic deuterium ions to obtain their energy distribution. We derive the S factor using the measured energy distribution of the ions, the measured volume of the fusion plasma,more » and the measured fusion yields. This method is model independent in the sense that no assumption on the state of the system is required, but it requires an accurate measurement of the ion energy distribution, especially at high energies, and of the relevant fusion yields. In the 2H(d,n)3He and 3He(d,p)4He cases discussed here, it is very important to apply the background subtraction for the energetic ions and to measure the fusion yields with high precision. While the available data on both ion distribution and fusion yields allow us to determine with good precision the S factor in the d+d case (lower Gamow energies), for the d+3He case the data are not precise enough to obtain the S factor using this method. Our results agree with other experiments within the experimental error, even though smaller values of the S factor were obtained. This might be due to the plasma environment differing from the beam target conditions in a conventional accelerator experiment.« less

  2. Henipavirus membrane fusion and viral entry.

    PubMed

    Aguilar, Hector C; Iorio, Ronald M

    2012-01-01

    Nipah (NiV) and Hendra (HeV) viruses cause cell-cell fusion (syncytia) in brain, lung, heart, and kidney tissues, leading to encephalitis, pneumonia, and often death. Membrane fusion is essential to both viral entry and virus-induced cell-cell fusion, a hallmark of henipavirus infections. Elucidiation of the mechanism(s) of membrane fusion is critical to understanding henipavirus pathobiology and has the potential to identify novel strategies for the development of antiviral therapeutic agents. Henipavirus membrane fusion requires the coordinated actions of the viral attachment (G) and fusion (F) glycoproteins. Current henipavirus fusion models posit that attachment of NiV or HeV G to its cell surface receptors releases F from its metastable pre-fusion conformation to mediate membrane fusion. The identification of ephrinB2 and ephrinB3 as henipavirus receptors has paved the way for recent advances in our understanding of henipavirus membrane fusion. These advances highlight mechanistic similarities and differences between membrane fusion for the henipavirus and other genera within the Paramyxoviridae family. Here, we review these mechanisms and the current gaps in our knowledge in the field.

  3. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

    PubMed Central

    Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591

  4. Molecular Mechanisms Regulating Cell Fusion and Heterokaryon Formation in Filamentous Fungi.

    PubMed

    Daskalov, Asen; Heller, Jens; Herzog, Stephanie; Fleißner, André; Glass, N Louise

    2017-03-01

    For the majority of fungal species, the somatic body of an individual is a network of interconnected cells sharing a common cytoplasm and organelles. This syncytial organization contributes to an efficient distribution of resources, energy, and biochemical signals. Cell fusion is a fundamental process for fungal development, colony establishment, and habitat exploitation and can occur between hyphal cells of an individual colony or between colonies of genetically distinct individuals. One outcome of cell fusion is the establishment of a stable heterokaryon, culminating in benefits for each individual via shared resources or being of critical importance for the sexual or parasexual cycle of many fungal species. However, a second outcome of cell fusion between genetically distinct strains is formation of unstable heterokaryons and the induction of a programmed cell death reaction in the heterokaryotic cells. This reaction of nonself rejection, which is termed heterokaryon (or vegetative) incompatibility, is widespread in the fungal kingdom and acts as a defense mechanism against genome exploitation and mycoparasitism. Here, we review the currently identified molecular players involved in the process of somatic cell fusion and its regulation in filamentous fungi. Thereafter, we summarize the knowledge of the molecular determinants and mechanism of heterokaryon incompatibility and place this phenomenon in the broader context of biotropic interactions and immunity.

  5. Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.

    PubMed

    Boland, Thomas; Mironov, Vladimir; Gutowska, Anna; Roth, Elisabeth A; Markwald, Roger R

    2003-06-01

    We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology.

  6. [Element distribution analysis of welded fusion zone by laser-induced breakdown spectroscopy].

    PubMed

    Yang, Chun; Zhang, Yong; Jia, Yun-Hai; Wang, Hai-Zhou

    2014-04-01

    Over the past decade there has been intense activity in the study and development of laser-induced breakdown spectroscopy (LIBS). As a new tool for surface microanalysis, it caused widespread in materials science because of the advantage of rapid and high sensitivity. In the present paper, the distribution of Ni, Mn, C and Si near weld fusion line was analyzed on two kinds of weld sample. Line scanning mode analysis was carried out by three different kinds of methods, namely laser-induced breakdown spectroscopy (LIBS), scanning electron microscope/energy dispersive spectrometer (SEM/EDS) and electron probe X-ray microanalyser (EPMA). The concentration variation trend of Ni and Mn acquired by LIBS is coincident with SEM/EDS and EPMA. The result shows that the content of Ni and Mn was significantly different between weld seam and base metal on both the samples. The content of Ni and Mn was much higher in weld seam than in base metal, and a sharp concentration gradient was analyzed in the fusion zone. According to the distribution of Ni and Mn, all the three methods got a similar value of welded fusion zone width. The concentration variation trend of C and Si acquired by LIBS is not coincident with SEM/EDS and EPMA. The concentration difference between weld seam and base metal was analyzed by LIBS, but had not by SEM/EDS and EPMA, because of the low concentration and slight difference. The concentration gradient of C and Si in fusion zone was shows clearly by LIBS. For higher sensitivity performance, LIBS is much more adapted to analyze low content element than SEM/EDS and EPMA.

  7. Acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology.

    PubMed

    Russell, Charles J

    2014-01-01

    Membrane fusion is not spontaneous. Therefore, enveloped viruses have evolved membrane-fusion mediating glycoproteins that, once activated, refold, and release energy that fuses viral and cellular membranes. The influenza A virus hemagglutinin (HA) protein is a prototypic structural class I viral fusion glycoprotein that, once primed by proteolytic cleavage, is activated by endosomal low pH to form a fusogenic "leash-in-grooves" hairpin structure. Low-pH induced HA protein refolding is an irreversible process, so acid exposure in the absence of a target membrane leads to virus inactivation. The HA proteins of diverse influenza virus subtypes isolated from a variety of species differ in their acid stabilities, or pH values at which irreversible HA protein conformational changes are triggered. Recently, efficient replication of highly pathogenic avian influenza (HPAI) viruses such as H5N1 in avian species has been associated with a relatively high HA activation pH. In contrast, a decrease in H5N1 HA activation pH has been shown to enhance replication and airborne transmission in mammals. Mutations that alter the acid stabilities of H1 and H3 HA proteins have also been discovered that influence the amantadine susceptibilities, replication rates, and pathogenicities of human influenza viruses. An understanding of the role of HA acid stability in influenza virus biology is expected to aid in identifying emerging viruses with increased pandemic potential and assist in developing live attenuated virus vaccines. Acid-induced HA protein activation, which has provided a paradigm for protein-mediated membrane fusion, is now identified as a novel determinant of influenza virus biology.

  8. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    PubMed

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  9. Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster

    PubMed Central

    2010-01-01

    Background In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined. Results Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype. Conclusions Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm. PMID:20701765

  10. Aluminium-induced changes of fusion pore properties attenuate prolactin secretion in rat pituitary lactotrophs.

    PubMed

    Calejo, A I; Jorgačevski, J; Silva, V S; Stenovec, M; Kreft, M; Gonçalves, P P; Zorec, R

    2012-01-10

    Hormone secretion is mediated by Ca(2+)-regulated exocytosis. The key step of this process consists of the merger of the vesicle and the plasma membranes, leading to the formation of a fusion pore. This is an aqueous channel through which molecules stored in the vesicle lumen exit into the extracellular space on stimulation. Here we studied the effect of sub-lethal dose of aluminium on prolactin secretion in isolated rat pituitary lactotrophs with an enzyme immunoassay and by monitoring electrophysiologically the interaction of a single vesicle with the plasma membrane in real time, by monitoring membrane capacitance. After 24-h exposure to sub-lethal AlCl(3) (30 μM), the secretion of prolactin was reduced by 14±8% and 46±11% under spontaneous and K(+)-stimulated conditions, respectively. The frequency of unitary exocytotic events, recorded by the high-resolution patch-clamp monitoring of membrane capacitance, a parameter linearly related to the membrane area, under spontaneous and stimulated conditions, was decreased in aluminium-treated cells. Moreover, while the fusion pore dwell-time was increased in the presence of aluminium, the fusion pore conductance, a measure of fusion pore diameter, was reduced, both under spontaneous and stimulated conditions. These results suggest that sub-lethal aluminium concentrations reduce prolactin secretion downstream of the stimulus secretion coupling by decreasing the frequency of unitary exocytotic events and by stabilizing the fusion pore diameter to a value smaller than prolactin molecule, thus preventing its discharge into the extracellular space. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Structural and functional properties of an unusual internal fusion peptide in a nonenveloped virus membrane fusion protein.

    PubMed

    Shmulevitz, Maya; Epand, Raquel F; Epand, Richard M; Duncan, Roy

    2004-03-01

    The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.

  12. Poxvirus entry and membrane fusion

    SciTech Connect

    Moss, Bernard . E-mail: bmoss@nih.gov

    2006-01-05

    The study of poxvirus entry and membrane fusion has been invigorated by new biochemical and microscopic findings that lead to the following conclusions: (1) the surface of the mature virion (MV), whether isolated from an infected cell or by disruption of the membrane wrapper of an extracellular virion, is comprised of a single lipid membrane embedded with non-glycosylated viral proteins; (2) the MV membrane fuses with the cell membrane, allowing the core to enter the cytoplasm and initiate gene expression; (3) fusion is mediated by a newly recognized group of viral protein components of the MV membrane, which are conserved in all members of the poxvirus family; (4) the latter MV entry/fusion proteins are required for cell to cell spread necessitating the disruption of the membrane wrapper of extracellular virions prior to fusion; and furthermore (5) the same group of MV entry/fusion proteins are required for virus-induced cell-cell fusion. Future research priorities include delineation of the roles of individual entry/fusion proteins and identification of cell receptors.

  13. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  14. Cell fusion as the formation mechanism of unreduced gametes in the gynogenetic diploid hybrid fish

    PubMed Central

    Wang, Jing; Liu, Qingfeng; Luo, Kaikun; Chen, Xuan; Xiao, Jun; Zhang, Chun; Tao, Min; Zhao, Rurong; Liu, Shaojun

    2016-01-01

    The gynogenetic diploid hybrid clone line (GDH) derived from red crucian carp (♀ RCC) × common carp (♂ CC) possesses the unusual reproductive trait of producing unreduced diploid eggs. To identify the mechanism underlying this phenomenon, we examined the structure, in vivo developmental process and in vitro dynamic development of the GDH gonad. In summary, compared with RCC and CC, GDH showed certain special straits. First, a high frequency (84.7%) of germ cell fusion occurred in gonadal tissue culture in vitro as observed by time-lapse microscopy. Second, microstructural and ultrastructural observation showed numerous binucleated and multinucleated germ cells in the gonad, providing evidence of germ cell fusion in vivo. By contrast, in the diploid RCC and CC ovaries, neither cell fusion nor multinucleated cells were observed during the development of gonads. Third, the ovary of GDH remained at stage I for 10 months, whereas those of RCC and CC remained at that stage for 2 months, indicating that the GDH germ cells underwent abnormal development before meiosis. This report is the first to demonstrate that cell fusion facilitates the formation of unreduced gametes in vertebrates, which is a valuable finding for both evolutionary biology and reproductive biology. PMID:27530321

  15. Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

    PubMed Central

    Schaal, H; Klein, M; Gehrmann, P; Adams, O; Scheid, A

    1995-01-01

    An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion

  16. Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells: results of a cell fusion experiment.

    PubMed

    Ozawa, Akio; Akasaka, Eri; Watanabe, Satoshi; Yoshida, Mitsutoshi; Miyoshi, Kazuchika; Sato, Masahiro

    2010-08-01

    Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells. In this study, we constructed a pOEIN plasmid carrying a ca. 5.4-kb mouse Oct-3/4 promoter linked to the EGFP cDNA and neomycin expression unit and produced a porcine embryonic cell line stably incorporating it in the genome. Cell fusion with mouse embryonal carcinoma cell line F9 resulted in generation of colonies with distinct EGFP-derived fluorescence around 14 days after fusion. RT-PCR using these colonies also confirmed expression of endogenous porcine pluripotency-specific Oct-3/4, Sox2 and Stat3 mRNA. These findings suggest that mouse-derived components are sufficient to induce dedifferentiation of differentiated pig cells and also that reprogramming proceeds gradually. The present non-invasive reporter system will be useful to better define the reprogramming mechanism and/or to identify novel reprogramming molecules in the pig.

  17. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.

    PubMed

    Rambold, Angelika S; Cohen, Sarah; Lippincott-Schwartz, Jennifer

    2015-03-23

    Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells.

  18. pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

    PubMed

    Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

    2017-06-01

    The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins.IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced

  19. Electro-acoustic fusion of erythrocytes and of myeloma cells.

    PubMed

    Vienken, J; Zimmermann, U; Zenner, H P; Coakley, W T; Gould, R K

    1985-11-07

    Mammalian cells can be concentrated in a sound field. A method is introduced, which combines the reversible aggregation of cells in a sound field with the electrical breakdown of cell membranes to fuse cells, which are in contact. Human red blood cells and mouse myeloma cells are fused by means of that procedure.

  20. Evaluation of EML4-ALK Fusion Proteins in Non-Small Cell