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[
Dev Cell,
2007]
Our understanding of the developmentally critical process of cell-cell fusion has been greatly advanced by the identification of the first family of cell-cell fusion proteins. Together, the two founding members of the FF family execute the majority of cell-cell fusion events in C. elegans.
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[
International Worm Meeting,
2007]
The fusion of cell membranes is fundamental in development and viral pathogenesis. In early development cell fusion is required for fertilization and is involved in stem cell trans-differentiation and organ formation, including the placenta, muscles and bones. Apart from normal development, cell fusion has been implicated in cancer metastasis. While viral cell fusion has been thoroughly characterized, the mechanism of developmental cell fusion remains purely understood. We identified EFF-1 a type I membrane protein necessary for most of the tissue specific cell fusion events in the nematode C. elegans and which can ectopically fuse cells both in vivo and in tissue culture (Podbilewicz et al., 2006). By comparing the amino acid sequence of EFF-1 using BLAST we have identified a novel EFF-1 related gene,
aff-1. AFF-1 is responsible for the fusion of the anchor cell to the utse syncytium and for fusion of the seam cells at the L4 stage (Sapir et al. 2007). AFF-1 shows 26% identity and 43% similarity to EFF-1, therefore these proteins constitute a novel family of developmental fusogens (FF Family). We have identified FF Family members in other nematodes and cloned the EFF-1 homologue in Pristionchus Pacificus showing 44% identity and 61% similarity to the C. elegans EFF-1. All members of the FF family share sixteen conserved cysteine residues in their ectodomains including a putative TGF-beta-type-I-Receptor-like domain. By analyzing the multiple sequence alignment of the known FF family members we have identified various domains that may be mechanistically important for fusion. We will discuss the effect of mutations on the FF fusion capacity and their possible mechanistic implications.
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Valansi, Clari, Raveh, Hadas, Verdin Ramos, Jorge, Avinoam, Ori, Smurova, Ksenia, Oren-Suissa, Maital, Podbilewicz, Benjamin, Friedlander, Lilach, Fridman, Karen
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Cell-cell fusion is essential for fertilization and organ development. Molecular genetic studies of cell fusion in C. elegans have led to the discovery of EFF-1 and AFF-1, founder members of the FF family of proteins that mediate cell membrane fusion. EFF-1 and AFF-1, are type I membrane glycoproteins that are required for cell fusion in eukaryotes. FF proteins can fuse cells when ectopically expressed on the plasma membranes of C. elegans and tissue culture cells. One third of all cells in C. elegans fuse homotypically and specifically either via EFF-1 or AFF-1. Cells can change shape by fusing with themselves through a process we call auto-cell fusion. FF proteins fuse cells via h emifusion, the universal intermediate in which the outer leaflets of the plasma membranes merge before the inner leaflets. The FF proteins are genuine cell fusogens because they are: (1) Essential for cell fusion. (2) Expressed specifically on the surface of both fusing plasma membranes at the time of fusion. (3) Sufficient to fuse cells that normally do not fuse in vivo and in heterologous tissue culture cells. We will show how FF fusogens sculpt epithelial toroidal cells, muscular rings, tubular membranes, and neuronal trees. We will also compare exoplasmic membrane fusion and tube formation with endoplasmic fusion and fission of organelles. Unveiling the mechanisms of cell fusion by EFF-1 and AFF-1 may facilitate the identification of other fusogens required for fertilization, neuronal dendritic arborization and somatic fusion across all kingdoms of life.
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[
International Worm Meeting,
2009]
Membrane fusion is the process by which membranous compartments unite to form a single compartment. Virtually all membranes can fuse, ranging from small intracellular vesicles and organelles to entire cells. Thus, membrane fusion is critical for many biological processes such as intracellular trafficking and exocytosis, fertilization and viral infection, embryonic and post embryonic development. With the exception of some viral fusogens, SNARE proteins, and FF proteins (AFF-1 and EFF-1), the large majority of fusogens remain unidentified or uncharacterized. The fusogenic activity of EFF-1 and AFF-1 was demonstrated by the finding that their ectopic expression in C. elegans and in Sf9 insect cells is sufficient to fuse cells that normally do not fuse. Fusion of heterologous cells by FF proteins indicates that they are bona fide fusogens. Thus, FFs can be used to understand the principles of eukaryotic cell-cell fusion machineries. Using sequence comparison of 18 nematode species, 31 FF putative orthologs were identified and analyzed. We found that FF proteins show similar domain architecture; all members are type I membrane proteins with an extracellular portion 520-540aa long and a variable cytoplasmic tail. In addition all members harbor the same regions of high conservation along with 16 conserved Cysteines in their extracellular portion. We expressed FFs from C. elegans and Trichinella spiralis, in Baby hamster kidney cells (BHK-21) and observed that the proteins are distributed both in intracellular compartments and at the cell surface. In addition, we determined that FF proteins from these species can fuse BHK-21 mammalian cells. Furthermore, expression of the Pristionchus pacificus EFF-1 ortholog in C. elegans embryos resulted in ectopic fusion. These results suggest that FFs are functionally conserved in nematodes, that they may be interchangeable between species, and that they can fuse insect and mammalian cells. It has been previously shown that expression of EFF-1 is required on both fusing membranes in order for them to fuse. Surprisingly, while this homotypic mode of action is conserved for AFF-1, expression of AFF-1 and EFF-1 leads to heterotypic fusion in tissue culture. Taken together these results suggest that FF proteins are folded in a similar three-dimensional structure which may be essential for their fusogenic activity. To test this hypothesis and to study the molecular mechanism of FF proteins we perform structure function analysis of AFF-1, we generated mutations and tested their effect on surface expression and protein function in tissue culture and in worms. We will discuss insights on the mechanism of AFF-1 and their evolutionary implications.
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[
Worm Breeder's Gazette,
1993]
THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878
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[
Nematologica,
1976]
The growth promoting activity of protein-haemin co-precipitates from ferritin, apoferritin, transferrin, bovine serum albumin, conalbumin and egg white on maturation and reproduction of C. briggsae has been evaluated. Ferritin, apoferritin and transferrin were found to be biologically highly active in the presence of haemin. Bovine serum albumin, conalbumin and egg white were slightly active. Maturation and reproduction of C. briggsae on the coagulates from bovine serum albumin and egg white were nearly independent of the dose administered, probably because the limited availability of haemin from these coagulates permits but slow growth, even in the presence of abundant proteinaceous material. Bovine serum albumin, egg white and conalbumin failed to support continuous growth of C. briggsae. It is supposed that the limited availability of haemin from these coagulates inhibits normal maturation and reproduction of the F1 progeny. These experiments clearly demonstrate the requirement for particulate haem. The requirement for
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[
Worm Breeder's Gazette,
1999]
For everyone who deals with the characterization of expression patterns in the nervous system, the truly impressive paper of White et al., 1986 ("Mind of the Worm") serves as the ultimate source of knowledge. While the largely invariant neural cell body positions described by Sulston et al. are an essential tool in the identification of neurons, the axon morphologies described by White et al. greatly facilitate the identification of a given neuron.
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[
International C. elegans Meeting,
1991]
The embryonic DD motoneurons eIhibit different target specificities at different stages of development (White, et al., Nature, 1978). John White has determined that the period of respecification is closely associated with the molt from Ll to L2 and simultaneous with the completion of morphological differentiation of the postembryonic VD motoneurons. Morphologically the two classes of D motoneurons are very similar, eacb having a process in the dorsal nerve cord, a process in the ventral cord, a connecting commissure and a ventrally positioned cell body. However they exhibit different patterns of synapses. Currently only one gene has been identified that affects one class of D neuron and not the other. Interstingly that gene, unc-ff, alters the pattern of synapses of the VD motoneurons so that it becomes identical to the DD pattern. Are the VD motoneurons in unc-ff mutants following the same sequence as described for the DD motoneurons (ie transiently forming one pattern of synapses and then switching)? To answer this question we are developing EMimmuno methods using antisera to the D motoneuron neurotransmitter GABA. Although we have no answer at this time, the technique does appear to be working and is of enough general applicability to warrant discussion. Worms, encased in agar, are fixed in I % glutaraldehyde and 4% paraformaldehyde. They are then dehydrated in alcohol and embedded in LR Gold resin and cold polymerized (4 for 16 hours (o/n)) under UV light. Tissue was processed according to procedures detailed in Okamoto and Thomson (J. Neurosci.1985). An antirabbit secondary antiserum conjugated tol O angstrom colloidal 801d partides makes it possible to view the antigen antibody complex at the EM level.
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[
Microsc Microanal,
2014]
A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.
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Valansi C, Zeev-Ben-Mordehai T, Grunewald K, White JM, Maurer UE, Avinoam O, Abutbul I, Fridman K, Podbilewicz B, Danino D, Sapir A
[
Science,
2011]
Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.