The type I membrane protein EFF-1 is essential for developmental cell fusion

Multinucleate cells are widespread in nature, yet the mechanism by which cells fuse their plasma membranes is poorly understood. To identify animal fusogens, we performed new screens for mutations that abolish cell fusion within tissues of C. elegans throughout development. We identified the gene eff-1, which is expressed as cells acquire fusion competence and encodes a novel integral membrane protein. EFF-1 sequence motifs suggest physicochemical actions that could cause adjacent bilayers to fuse. Mutations in the extracellular domain of EFF-1 completely block epithelial cell membrane fusion without affecting other perfusion events such as cell generation, patterning, differentiation, and adhesion. Thus, EFF-1 is a key component in the mechanism of cell fusion, a process essential to normal animal development.     

Cell fusion: an EFFicient sculptor

Mutations in the eff-1 gene of Caenorhabditis elegans, which prevent all cell-cell fusions in the nematode's epidermis, have revealed developmental roles for cell fusion. An extracellular fusogen-like domain in EFF-1 suggests it might direct the fusion of lipid bilayers.     

The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.     

N-ethylmaleimide sensitive factor is required for fusion of the C. elegans uterine anchor cell

The fusion of the Caenorhabditis elegans uterine anchor cell (AC) with the uterine-seam cell (utse) is an excellent model system for studying cell-cell fusion, which is essential to animal development. We obtained an egg-laying defective (Egl) mutant in which the AC fails to fuse with the utse. This defect is highly specific: other aspects of utse development and other cell fusions appear to occur normally. We find that defect is due to a missense mutation in the nsf-1 gene, which encodes N-ethylmaleimide-sensitive factor (NSF), an intracellular membrane fusion factor. There are two NSF-1 isoforms, which are expressed in distinct tissues through two separate promoters. NSF-1L is expressed in the uterus, including the AC. We find that nsf-1 is required cell-autonomously in the AC for its fusion with the utse. Our results establish AC fusion as a paradigm for studying cell fusion at single cell resolution and demonstrate that the NSF ATPase is a key player in this process.     

ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions.     

Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans

BACKGROUND: Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS: We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS: EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.     

Repression of cell-cell fusion by components of the C. elegans vacuolar ATPase complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

ceh-16/engrailed patterns the embryonic epidermis of Caenorhabditis elegans

engrailed is a homeobox gene essential for developmental functions such as differentiation of cell populations and the onset of compartment boundaries in arthropods and vertebrates. We present the first functional study on engrailed in an unsegmented animal: the nematode Caenorhabditis elegans. In the developing worm embryo, ceh-16/engrailed is predominantly expressed in one bilateral row of epidermal cells (the seam cells). We show that ceh-16/engrailed primes a specification cascade through three mechanisms: (1) it suppresses fusion between seam cells and other epidermal cells by repressing eff-1/fusogen expression; (2) it triggers the differentiation of the seam cells through different factors, including the GATA factor elt-5; and (3) it segregates the seam cells into a distinct lateral cellular compartment, repressing cell migration toward dorsal and ventral compartments.     

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.     

Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis.

BACKGROUND: During cytokinesis, the plasma membrane of the parent cell is resolved into the two plasma membranes of the daughter cells. Membrane fusion events mediated by the machinery that participates in intracellular vesicle trafficking might contribute to this process. Two classes of molecules that are required for membrane fusion are the t-SNAREs and the v-SNAREs. The t-SNAREs (syntaxins) comprise a multi-gene family that has been suggested to mediate, at least in part, selective membrane fusion events in the cell. RESULTS: We have analyzed the genome of Caenorhabditis elegans and identified eight syntaxin genes. RNA-mediated interference (RNAi) was used to produce embryos deficient in individual syntaxins and these embryos were phenotypically characterized. Embryos deficient in one syntaxin, Syn-4, became multinucleate because of defects in karyomere fusion and cytokinesis. Syn-4 localized both to ingressing cleavage furrows and to punctate structures surrounding nuclei as they reformed during interphase. CONCLUSIONS: Our analyses indicate that both cytokinesis and reformation of the nuclear envelope are dependent on SNARE-mediated membrane fusion.     

REF-1, a protein with two bHLH domains, alters the pattern of cell fusion in C. elegans by regulating Hox protein activity

Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. Here we identify a gene, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis.     

Targeting of rough endoplasmic reticulum membrane proteins and ribosomes in invertebrate neurons

The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins.     

Expression cloning of oligomerization-activated genes with cell-proliferating potency by pseudotype retrovirus vector

We developed a method of clone proliferation promoting fusion genes whose proteins were activated by protein oligomerization through the helix-loop-helix region (PNT domain) of TEL. We inserted a cDNA library downstream of the PNT domain with a retrovirus vector. The resulting retrovirus infected cytokine-dependent 32D cells and cells with cytokine-independent growth were analyzed for the inserted cDNA. We cloned 25 independent fusion genes including seven kinds of partner genes. Six of the seven were a fusion of TEL with protein tyrosine kinase, LYN, HCK, FGR, SYK, FLT3, and TYK2. A serine/threonine kinase, ARAF1, was also found to fuse with TEL. These kinase fusion proteins included kinase domains with proper reading frames. These fusions may be a useful model for clarifying the downstream signal transduction of constitutive active kinase and this expression cloning method may provide a new tool with which to study cell proliferation signalling.     

Role of microtubules in fusion of post-Golgi vesicles to the plasma membrane

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.