Cell-cell fusion is critical for the conception, development, and physiology of multicellular organisms. Although cellular fusogenic proteins and the actin cytoskeleton are implicated in cell-cell fusion, it remains unclear whether and how they coordinate to promote plasma membrane fusion. We reconstituted a high-efficiency, inducible cell fusion culture system in the normally nonfusing Drosophila S2R+ cells. Both fusogenic proteins and actin cytoskeletal rearrangements were necessary for cell fusion, and in combination they were sufficient to impart fusion competence. Localized actin polymerization triggered by specific cell-cell or cell-matrix adhesion molecules propelled invasive cell membrane protrusions, which in turn promoted fusogenic protein engagement and plasma membrane fusion. This de novo cell fusion culture system reveals a general role for actin-propelledinvasive membrane protrusions in driving fusogenic protein engagement during cell-cell fusion.. ...
Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell-cell contact where fusion occurs. In matings between Deltaprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by ...
Common themes are emerging from the study of viral, cell-cell, intracellular, and liposome fusion. Viral and cellular membrane fusion events are mediated by fusion proteins or fusion machines. Viral fusion proteins share important characteristics, notably a fusion peptide within a transmembrane-anchored polypeptide chain. At least one protein involved in a cell-cell fusion reaction resembles viral fusion proteins. Components of intracellular fusion machines are utilized in multiple membrane trafficking events and are conserved through evolution. Fusion pores develop during and intracellular fusion events suggesting similar mechanisms for many, if not all, fusion events. ...
Experimental evidence points towards a remarkably conserved mechanism by which virally encoded envelope glycoproteins catalyse membrane fusion and facilitate delivery of the viral core into the target cell [13, 14]. The structures of several class 1 fusion proteins reveal a characteristic "trimer-of-hairpins" motif believed to represent a late or post-fusion conformation [16-19, 35-37]. Investigating the way in which envelope proteins fold from a rod-like, pre-hairpin intermediate into the trimer-of-hairpins to pull the viral and cellular membranes together is important not only for our understanding of viral entry but also for the development of therapeutically relevant inhibitors of this process.. The protein sequences of the TM ectodomains of BLV and HTLV-1 display a striking level of conservation. By scrutinizing the position of conserved residues in the context of the HTLV-1 six-helix-bundle structure, we have found that the majority of the conserved residues map to the interacting surfaces ...
SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution bef
Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by
Several viral envelope glycoprotein oligomers assembled into a viral fusion machine, form a molecular scaffold that brings the viral and target cell membranes into close apposition and allow the subsequent fusion events. The fusion pore formation and its sequential expansion are orchestrated by viral and cellular lipids and proteins. The HIV entry process is understood in some detail at the molecular level. It is coordinated by the HIV envelope glycoprotein complex, a trimer of three gp120 surface glycoproteins, each noncovalently attached to three gp41 ransmembrane glycoprotein subunits.%&/It is know that changes in GSLs expression in target membranes can modulate viral fusion and entry. These studies on structure-function relationship of target membrane GSLs, the gp120-gp41 and the viral receptors suggest that plasma membrane GSLs support HIV-1 entry by stabilizing the intermediate steps in the fusion cascade. These observations, led it to hypothesize that upregulation of GSLs metabolites ...
The vacuole system provides good tools to assay the abundance of the tagged SNAREs on the isolated organelle, dissect their molecular interactions, and to identify hemifusion intermediates. In contrast to previous studies on tagged synaptobrevin II in chromaffin cells, in which these molecular properties are hard to access [40], this allowed us to demonstrate that the effect of large luminal tags was restricted to content mixing, whereas lipid mixing was essentially unaffected. This suggests that mixing of the outer leaflets may be less dependent on a collective perturbation of lipid structure by SNARE TMDs than the rearrangement of the inner leaflets. It is consistent with theory and simulations on the energetics of SNARE‐driven fusion, which suggested that fusion pore opening is limited by a larger free energy barrier than the induction of hemifusion [38]. In line with this, opening of the fusion pore has been found to be rate‐limiting for vacuole fusion [22].. The observation that even ...
An analysis of the R18 fusion assay was made during the fusion of the Sendai virus with erythrocyte ghosts. The increase in R18 fluorescence, reflecting the interaction process, was evaluated in terms of the different processes that in principle may contribute to this increase, that is, monomeric probe transfer, hemifusion, and complete fusion. To this end, the kinetics of the R18-labeled lipid mixing were compared to those obtained with an assay in which the fusion-monitoring probe, eosin-maleimide, was attached to the viral surface proteins. The experiments relied on the use of native and fusion-inactive viruses and studies involving viral and target membranes that were modified by the incorporation of the lysophospholipid. The total dequenching signal detected in the R18 assay consists of components from probe transferred without fusion and from fusion itself. At 37 degrees C, the initial rate of dequenching (within two minutes) was predominately from the probe diluted by fusion with little
Role of the synaptobrevin C terminus in fusion pore formation.: Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known a
Using the well-established, stage-specific model of fully primed, fusion-ready CVs (Coorssen et al., 1998; Coorssen et al., 2003; Hibbert et al., 2005; Churchward et al., 2005), we find that raft integrity underlies the efficiency of fast, Ca2+-triggered native membrane fusion, but not the fundamental ability to fuse. Using selective methods, including enzymatic digestion and sequestration within the membrane (Churchward et al., 2005), we show that the integrity of microdomains rich in SM-CHOL correlates directly with Ca2+ sensitivity and late fusion kinetics. In terms of the Ca2+ sensitivity of triggered fusion, SM does not appear to contribute directly, but rather through its role as a microdomain organizer. Thus, unlike its neighboring CHOL molecules, SM is not an essential component of the minimal native fusion machine. As a microdomain organizer, SM is associated with Ca2+ sensing and/or the interaction of additional proteinaceous or lipidic components that support the physiological Ca2+ ...
Author: Ngatchou, A. N. et al.; Genre: Journal Article; Published in Print: 2010-10-26; Title: Role of the synaptobrevin C terminus in fusion pore formation.
Single-particle studies of dengue-virus membrane fusion and the effect of small-molecule inhibitors of infection clarify the viral fusion mechanism.
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E1 is a class II viral fusion protein. This trimeric (low-pH-iduced) form is fusion active, and promotes release of viral nucleocapsid in cytoplasm after cell and viral membrane fusion. Efficient fusion requires the presence of cholesterol and sphingolipid in the target membrane. N-terminal domain of this protein: 1dyl(NMR), 1vcp, 1vcq ...
The SNARE hypothesis states that the folding and assembly of four-helix SNARE complex bundles drives intracellular membrane fusion, including fusion in exocytos...
Regulated exocytosis is a stimulus-dependent membrane fusion event of fundamental importance to a range of physiological processes. The membrane fusion reaction...
The possibility that isomerization controls the fusion activity was tested by analysing Mo‐MLV fusion and infectivity under conditions that either inhibited or induced isomerization. The fusion was studied as virus‐induced polykaryon formation in XC cells (fusion‐from‐without). Fusion of cell‐bound virus is induced by incubation at 37°C and terminated by pH 3.0 treatment. In confluent cultures (Figure 6A), the fusion will merge cells, and with time these will rearrange into polykaryons (Figure 6B). Preliminary testing demonstrated that TN/1.8 mM Ca2+ supported fusion as effectively as DMEM (data not shown). Therefore, TN/1.8 mM Ca2+ was used as the control condition. The time course of the fusion process is shown in Figure 6C.. We first studied the effect that alkylation‐mediated inhibition of isomerization had on fusion. To avoid adverse effects due to alkylation of internal viral proteins, we used the membrane‐impermeant reagents M135 and MTSET. We observed a ...
[BioChemistry] Vaccinia A27 Protein Structure is Revealed to Regulate Virus and Host Cell Membrane Fusion (Chinese Version) Academia Sinica Newsletter (2013/08/27) Two research teams in Academia Sinica, Dr. Andrew H.-J.
Alphaviruses, single-stranded RNA viruses within the Togaviridae family, are important human and animal pathogens. These viruses invade the host cells through the receptor-mediated endocytosis pathway. The acidic environment in the endosome induces fusion of the viral envelope and the endosomal membrane, allowing delivery of the viral genomic material into the cytoplasm of the infected cell. The energy cost for merging the hydrated membranes is endowed with the conformational changes and oligomeric rearrangements of the viral E1 glycoproteins, a class-II fusion protein. Although the crystal structures of the E1 ecto-domain in pre- and post-fusion conformations were determined, the structural details of the intermediate organizations of the fusion protein during the course of membrane fusion are poorly understood. Major obstacles that have impeded vigorous structural studies are the aggregation and heterogeneity of virus particles at low-pH, and additive heterogeneity introduced by non-uniform ...
Fusion pore regulation of transmitter release.: During the last decade a wealth of new information about the properties of the exocytotic fusion pore is changin
Here are some pics that will show how far I can twist with a T2-pelvic fusion. I also have pics of my rib hump, which is now much less. I still have about 30 degrees rotation in my vert, verified by my cts. My hump was much worse before my surgeries. Hopes this gives some idea as to limitations after a full fusion. Ed
Visualizing the location and dynamics of exocytosis Toomre et al. use a combination of TIR microscopy (green, labeling molecules close to or at the membrane) and standard fluorescence microscopy (red, for molecules further from the membrane) to visualize trafficking to and fusion with the plasma membrane during exocytosis. Red dots turn yellow then green as they approach the membrane, and then explode in a burst of light as they fuse with the plasma membrane during exocytosis. The transport containers appear to be partially anchored at the membrane before fusion, and can undergo either partial or complete fusion events ...
Antiviral blocking peptides targeting the viral fusion core can inhibit viral membrane fusion, thereby inhibiting the viruss entry into the host cell.
Description Recombinant SARS-COV-2 S1+S2 ECD(S-ECD) Protein is produced by HEK293 cells expression system. The target protein is expressed with sequence (Val11-Gln1208) of SARS-COV-2 S1+S2 ECD(S-ECD) (Accession...
The IR-110 A is the new generation of infrared fusion machines. Automation and intuitive handling enable highest efficiency for installers and operators.
The IR-110 A is the new generation of infrared fusion machines. Automation and intuitive handling enable highest efficiency for installers and operators.
hardware after fusion - MedHelps hardware after fusion Center for Information, Symptoms, Resources, Treatments and Tools for hardware after fusion. Find hardware after fusion information, treatments for hardware after fusion and hardware after fusion symptoms.
A workshop on fusion technology beyond ITER was successfully held between the Japanese and the Korean Domestic Agencies on 8-9 November at the National Fusion [...]
4I78: Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities.
Aguilar, P.S., Baylies, M.K., Fleissner, A., Helming, L., Inoue, N., Podbilewicz, B., Wang, H., and Wong, M. (2013). Genetic basis of cell-cell fusion mechanisms. Trends in genetics : TIG 29, 427-437 ...
Provides in-depth assessment of 500+ genes associated with fusions in cancer, including solid tumors, soft tissue cancers and hematological malignancies. Delivers detected fusions in a simple report.
For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein seq ...
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Oracle Retail Predictive Application Server - Version 16.0.1 and laterRPAS Fusion Client: Changing a Value in Cell with Real Time Alert Throws Error Cannot read pro
The really bad thing is that it looks like the disc above my fusion is poking into the cord. There was very little space and at on point it seemed like there was no space. There was another disk below the fusion that looked questionable but not as bad as the one above it ...
The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane ...
As for most cell-cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca(2+) influx, yields similar cell fusion defects. Although extracellular Ca(2+) is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca(2+) is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca(2+) binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed ...
The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söll …
Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: A multifunctional quantitative assay for biotins profile, publications, research topics, and co-authors
New 3-D maps of water distribution during cellular membrane fusion are accelerating scientific understanding of cell development, which could lead to new treatments for diseases associated with cell fusion. Using neutron diffraction at the Department of Energys Oak Ridge National Laboratory, researchers have made the first direct observations of water in lipid bilayers used to model cell membrane fusion.
Our studies show that vacuole-bound actin is needed for homotypic fusion of this organelle in the absence of cytoskeleton or cytosol. Proteins of the well-established pathways of actin cytoskeleton regulation are needed for normal vacuole structure in vivo (Fig. 2 A) and are found on purified vacuoles at levels which cannot be due to cytosolic contamination (Fig. 2 B). Antibody to the Las17p/Bee1p, the yeast WASp homologue, inhibits vacuole fusion (Fig. 3), and this inhibition can be modulated (Fig. 3 C) by high levels of either the WCA domain of Las17p or by calmodulin, which are known to interact directly with Arp2/3 complex. Antibody to Arp3p itself also blocks vacuole fusion (Fig. 3 D). Mutations in actin have striking effects on vacuole structure in vivo (Fig. 2 A) and fusion in vitro (Fig. 4), and well-studied actin ligands (Morton et al., 2000) show fusion stage-specific inhibition of the vacuole fusion reaction (Figs. 5 and 6). We found that blocking F-actin depolymerization ...
Maintenance of eukaryotic cellular homeostasis requires the fusion of vesicle membranes that is accomplished by a SNARE-mediated mechanism. Membrane fusion is the merger of two lipid bilayers into one continuous membrane. Multiprotein complexes that have been conserved in eukaryotes carry out the basic reactions of fusion. In Saccharomyces cerevisiae, homotypic vacuole fusion occurs in experimentally defined phases. Fusion priming does not involve contact between vacuoles but includes the disassembly of complexes of SNAREs on the same membrane (cis) by Sec18p (NSF) and its cochaperone Sec17p (a-SNAP). Tethering requires Ypt7p (a Rab GTPase) and the HOPS effecter complex. SNARE complexes, including one R SNARE from a donor vacuole and three Q SNAREs from the acceptor vacuole, are formed in trans during docking of vacuoles. The membranes of the docked vacuoles are drawn together to form the "boundary domain" that resembles flat discs. The outer membranes are not in contact and come together at the ...
For the 15 years or so, we have developed a series of reconstitution platforms to investigate the molecular mechanism of SNARE-mediated membrane fusion, and the regulation of this process by regulatory factors. In 1998, we established the central function of the SNARE proteins as fusogens when we reconstituted these proteins into small unilamellar vesicle (SUV) and measured lipid mixing between liposomes containing cognate SNARE proteins.. We have since demonstrated SNARE-mediated fusion by reconstituting SNAREs into giant unilamellar vesicle (GUV, shown in Figure 1) and supported bilayer (SBL, shown in Figure 2). Both of the systems provided flat, and therefore, more physiologically relevant membrane environment on the t-SNARE side. In the SUV-SBL fusion system, we have measured single fusion event in millisecond time-scales.. ...
Membrane fusion, the merger of two biological membranes without content leakage, is essential for protein transport along the exocytic and endocytic pathways in eukaryotic cells. Fusion requires conserved membrane‐anchored proteins named SNAREs (αSNAP receptors) (Wickner and Schekman, 2008; Sudhof and Rothman, 2009), which reside on both the donor and acceptor membranes. SNAREs form four helical coiled‐coil bundles through their heptad‐repeat SNARE domains. Cis‐SNARE complexes are disassembled by Sec17p/αSNAP and Sec18p/NSF in an ATP‐dependent step called priming. Liberated SNAREs from apposed membranes form trans‐SNARE complexes, an essential step for fusion. Other proteins cooperate with the SNAREs to achieve fusion. Rab GTPases and their effectors promote the tethering of donor and acceptor membranes (Grosshans et al, 2006; Markgraf et al, 2007; Hickey and Wickner, 2010) and thereby indirectly promote trans‐SNARE complex formation. Sec1p/Munc18 (SM) proteins bind individual ...
Lysolipids added between fusing membranes inhibit and cis-unsaturated fatty acids promote not only diverse biological fusion reactions (this paper and Creutz, 1981; Glick and Rothman, 1987; Chernomordik et al., 1993; Paiement et al., 1994; Yeagle et al., 1994; Chernomordik et al., 1995c; Gunther-Ausborn et al., 1995; but see Nagao et al., 1995; Coorssen, 1996), but also fusion of purely lipid bilayers (for review see Chernomordik et al., 1995b). Importantly, LPC inhibits HA-mediated fusion at membrane concentrations similar to those found to inhibit syncytia formation mediated by the Sendai virus F protein (Yeagle et al., 1994) and baculovirus gp64 (Chernomordik et al., 1995c), as well as for microsome-microsome fusion (Chernomordik et al., 1993) and vesicle-planar bilayer fusion (Chernomordik et al., 1995a). We suggest that fusion mediated by HA and other proteins and fusion of purely lipid bilayers proceed via a common lipid-involving intermediate-a stalk structure, producing local and ...
Vesicle associated membrane proteins are members of the soluble N-ethyl-maleimide-sensitive factor receptor (SNARE) attachment protein family that facilitate the intracellular membrane fusion process involved in neurotransmitter release (Ungar & Hughson, 2003). Cellubrevin, or VAMP-3, is similar to SNARE proteins, synaptobrevin 1 and 2 (VAMP-1 and 2), but can be found in many different tissues such as fibroblasts, adipose cells, insulin-secreting B cells, and supportive brain cells like glial, but not brain neuron cells (Chilcote et al., 1995). However cellubrevin is also a substrate for proteolytic action of tetanus toxin just like VAMP-1 and 2, and suggests similar roles in exocytosis due to the blocking of neurotransmitter release in the presence of tetanus (Chilcote et al., 1995). Cellubrevin has been found to be significant in the docking and vesicle fusion process of secretory granules to the plasma membrane, but few studies in the immunofluorescence localization on subcellular fractions ...
2KXA: The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface.
Our approach described here provides a general avenue for observing single-liposome fusion events in proteoliposome systems (9, 12, 15, 34-36). Modification of the more conventional bulk-phase assays was kept minimal; one type of proteoliposome was attached to a nonsticky surface via specific interaction. Comprehensive controls and calibrations demonstrated that in vitro fusion activity in bulk solution is preserved in single-liposome fusion on surface. Real-time monitoring of SNARE-mediated, single-liposome fusion has revealed several key features: existence of hemifusion and additional intermediates on the pathway to full fusion and kinetic information on individual intermediate states. Furthermore, our assay might enable the dissection of the different fates of liposomes after fusion, for example, kiss-and-run type detachment. We should, however, emphasize that our work is based on yeast SNAREs and with a relatively high protein-to-lipid ratio (1:100) and therefore does not yet address the ...