White JM (2007) Dev Cell "The first family of cell-cell fusion."

White JM

[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.

Ori Avinoam et al. (2007) International Worm Meeting "How Do They Fuse it? Introducing the FF Family."

Amir Sapir, Ori Avinoam, Benjamin Podbilewicz

[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.

Podbilewicz, Benjamin et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "How EFF-1 and AFF-1 fuse cell membranes and sculpt epithelia, muscles and neurons"

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.

Avinoam, Ori et al. (2009) International Worm Meeting "Evolutionary Conservation of Cell Fusion in Nematodes."

Avinoam, Ori, Podbilewicz, Benjamin

[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.

Avinoam O et al. (2011) Science "Conserved eukaryotic fusogens can fuse viral envelopes to cells."

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.

Favaro PM et al. (2003) Biochem Biophys Res Commun "Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt."

de Souza Medina S, Basseres DS, Saad ST, Favaro PM, Costa FF, Traina F

[Biochem Biophys Res Commun, 2003]

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.

Amir Sapir et al. (2008) Development & Evolution Meeting "EFF-1 and AFF-1, Founders of a Family of Cell-Cell Fusion Proteins in Nematodes"

Meital Oren-Suissa, Amir Sapir, Ori Avinoam, Clari Valansi, Benjamin Podbilewicz

[Development & Evolution Meeting, 2008]

Cell fusion is essential for sexual reproduction and organ development. Yet, despite many years of research, little is known about the mechanism of cell-cell fusion. Our molecular genetic studies of developmental cell fusion in C. elegans have led to the discovery of the first family of proteins that mediate cell fusion (the FF family of fusogens). These fusogens, EFF-1 and AFF-1, are type I membrane proteins that are essential for cell fusion and can fuse cells when ectopically expressed on the membranes of C. elegans and heterologous insect or mammalian cells. EFF-1 is a developmental fusogen that drives most epithelial fusion events in C. elegans. AFF-1 is responsible for the last step in the life of the anchor-cell: its fusion to the utse syncytium forming the worm hymen. AFF-1 is necessary to fuse the anchor-cell, hyp5, a few pharyngeal muscles, the vulA and vulD rings of the vulva, and the lateral seam cells. We suggest that there is a structural domain shared between AFF-1, EFF-1 and TGF-beta-type-I-receptors. Furthermore, FOS-1 controls AFF-1-mediated developmental anchor-cell fusion. We will discuss how the FF proteins fuse cells via an universal intermediate in which the outer leaflets of the plasma membranes merge before the inner leaflets (hemifusion). The FF proteins are the first genuine cell fusogens because they are: (1) Essential for mesodermal and ectodermal cell-cell fusion. (2) Expressed on the surface of both fusing cells at the time of cell fusion. (3) Sufficient to fuse cells that normally do not fuse in vivo and in tissue culture cells. Understanding the mechanism of cell fusion mediated by EFF-1 and AFF-1 may facilitate the identification of other fusogens required for fertilization and somatic fusion in different organisms.

Rose AM et al. (1983) Worm Breeder's Gazette "RFD's as probes for cloning."

Beckenbach KA, Rose AM, Mawji NR, Baillie DL, Nelson DW

[Worm Breeder's Gazette, 1983]

We have been screening for restriction fragment differences (RFDs) between the N2 and Bo strains as described by Emmons, et al., 1979. Random N2 fragments with EcoRI and HindIII ends were cloned into pBR322, isolated, and purified. Using these fragments as P nick translated probes, RFDs were detected about one per six tested probes. We are in the process now of accumulating and mapping a set of these probes. It is our hope to establish a reasonably large collection of mapped RFD probes that might be utilized in the cloning of genes with no biochemically identified products. Towards this end we have mapped four such RFDs: One of these pGe s18 has been further mapped to the left of unc-13 ( I). Terry Snutch has screened our Charon 4 library for corresponding lambda clones of this region, which we have restriction mapped and subcloned for chromosome walking. We hope that characterization of the DNA adjacent to pCe s18 will allow us to isolate some of the genes in the unc-15 region described by Rose and Baillie, 1980. Interestingly, probe pCe s18 when hybridized against N2, Bo, and FF shows the N2 banding pattern. We would welcome information on the relationship (if any) between Bo and FF. [See Figure 1]

Gattegno T et al. (1997) International C. elegans Meeting "A MUTATION AFFECTING CELL FUSIONS DURING ELONGATION OF THE EMBRYO"

Gattegno T, Podbilewicz B

[International C. elegans Meeting, 1997]

Cell fusion is a widespread process throughout development and evolution. Many organs in multicellular organisms contain multinucleate cells formed by cell fusion; e.g., in humans (placenta, bone and muscle) and in C. elegans (hypodermis, vulva, pharynx, uterus, and excretory gland). Enveloped viruses (e.g. HIV) fuse with cell membranes in order to invade target cells, and fertilization involves sperm-egg fusion. We have characterized the order of epithelial cell fusions in C. elegans (1) and have isolated mutations that disrupt cell fusions in the embryo. In our pilot screens we did not obtain any mutation with fewer fusions than in wild-type. The zygotic lethal mutation zu316 was isolated in a screen for mutations that arrest during embryonic elongation (2). In the strain JJ825 zu316/+; lin-2 (e1309) the dorsal hypodermal cells fail to fuse. This mutant was outcrossed and we are now characterizing zu316 by 4D microscopy and immunofluorescence using tissue-specific mAbs and confocal microscopy. The development of the mutant terminates between 1.5 to 2-fold stages of elongation but some embryos arrest earlier. The mutants appear normal up to the beginning of morphogenesis and then the dorsal hypodermal cells look round during elongation and do not get flattened as in N2. Using MH27 and anti-LIN26 antibodies (kindly provided by R. Waterston and M. Labouesse, respectively) we notice that in zu316 there are fewer cell fusions in the dorsal hyp6 and hyp7 than in wild-type embryos. 1. Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424 2. Costa M. and Priess J. (1995) Worm Meeting Abstract, p. 165 We thank Mike Costa and Jim Priess for sending JJ825 to us.

Gottschalk, Alexander et al. (2019) International Worm Meeting "Synapsin is required for cAMP-dependent neuropeptide release."

Shao, Jiajie, Yu, Szi-Chieh, Steuer Costa, Wagner, Liewald, Jana, Gottschalk, Alexander

[International Worm Meeting, 2019]

Release of neuropeptide-containing dense core vesicles (DCVs) is an important process in neuromodulation and in the switching of brain states. By optogenetics, behavioral studies, electrophysiology, electron microscopy, and imaging, we previously showed (Steuer Costa et al., 2017) that cAMP increase in cholinergic motor neurons leads to mobilization and increased fusion of synaptic vesicles (SVs), as well as DCVs. The neuropeptides released caused an auto-regulatory increase in SV filling with acetylcholine, observable by visibly increased SV diameter and increased miniature post-synaptic current (mPSC) amplitudes. Here, we show that synapsin is required for the cAMP-dependent stimulation of neuropeptide release. Synapsin is a phosphoprotein that tethers SVs to the actin cytoskeleton, and forms liquid-liquid phase-separated SV clusters via its C-terminal intrinsically disordered region. In synapsin mutants, DCVs are largely absent from synaptic terminals, and are not released following light-induced cAMP increase. While SV release still occurs, mPSC amplitudes are not increased, and no increase in SV diameter is observed in synapsin mutants. Synapsin mutant terminals are largely devoid of DCVs, which instead accumulate in the neuronal processes and cell bodies. We also analyzed CRISPR-engineered mutants of the major PKA phosphorylation site in synapsin SNN-1B, S9. The non-phosphorylatable S9A mutant was essentially normal, while the phosphomimetic S9E was affected for DCV release, and accumulated DCVs in synapse-distal regions of the neurons. Thus, synapsin captures DCVs at terminals, making them available for release. We are currently analyzing DCV trafficking in the mutant neurons. Steuer Costa, Yu, Liewald, Gottschalk, 2017, Curr Biol 27: 495-507