Final Report Summary - ELEGANSFUSION (Mechanisms of cell fusion in eukaryotes) Cell-cell fusion is a universal process necessary for sexual reproduction (e.g. mating and fertilization) and organ formation (e.g. muscles, bones, eye lens, and placenta). There are only two eukaryotic cell fusion families that have been shown to be both essential and sufficient for somatic cell-cell fusion: the Syncytins that fuse the syncytial trophoblast in placental animals and the FF (fusion family) that fuse one third of all cells generated in C. elegans. During organogenesis FF proteins mediate cell-cell fusion of somatic cells to sculpt giant cells into organs, such as the vulva, uterus, pharynx and skin in C. elegans (Weinstein and Podbilewicz, 2016). In contrast, there is no family of fusion proteins that have been shown to be both essential and sufficient for gamete fusion during sexual reproduction (Aguilar et al., 2013; Podbilewicz, 2014; Podbilewicz and Chernomordik, 2016).The main goals of ELEGANSFUSION were (1) to determine the physicochemical mechanism of cell-cell fusion mediated by EFF-1 and AFF-1; and (2) to find missing fusion proteins that act in diverse cell fusion events across all kingdoms of life.Our hypothesis-driven research proposed that eukaryotic exoplasmic fusion proteins (fusogens) from the FF family (e.g. EFF-1 and AFF-1 from C. elegans) fuse membranes using a mechanism analogous to the one used by well-established viral or endoplasmic fusogens. In a second working hypothesis we proposed that unidentified fusogens will fuse cells during sexual and somatic processes using same principles as FF proteins.We found that the ectodomain of EFF-1 crystal structure revealed a very high similarity to class II fusion proteins from enveloped viruses (e.g. zika, dengue and rubella). However, although the structure and function of the viral and somatic cell fusogens are practically the same, our experiments using C. elegans, mammalian cultured cells, and pseudotyped viruses we found that the mechanisms are very different (Avinoam, 2012; Avinoam et al., 2011; Avinoam and Podbilewicz, 2011; Pérez-Vargas et al., 2014). Using electron microscopy techniques we studied the structure of EFF-1 and AFF-1 on the membranes and determined that the protein is active when present on heterologous systems including mammalian cells, pseudotyped viruses and proteoliposomes (Avinoam, 2012; Avinoam et al., 2011; Avinoam and Podbilewicz, 2011; Fridman, 2012; Podbilewicz, in preparation).HAP2/GCS1 are type I membrane glycoproteins essential for gamete fusion in plants, Chlamydomonas, Plasmodium, Tetrahymena, and Dyctyostelium. To determine whether HAP2/GCS1 is not only essential but also sufficient for cell-cell fusion, we decided to express the Arabidopsis sperm HAP2/GCS1 in mammalian cells that normally do not fuse. We found that expression of HAP2/GCS1 results in fusion of mammalian cells. We also showed that HAP2/GCS1 has to be present in both fusing cells for plasma membrane merger to occur. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in HAP2-dependent virus cell fusion. Structural modeling of the HAP2/GCS1 protein family predicts that it is homologous to EFF-1 from C. elegans and class II viral fusion proteins (e.g. dengue, Zika, Rubella viruses). Remarkably, cell-cell fusion also occurs between EFF-1- and HAP2-expressing cells indicating that an animal somatic cell fusion protein (EFF-1) and a plant sperm fusion protein (HAP2/GCS1) can functionally interact. Moreover, we found that HAP2/GCS1 fusion is mediated via a universal hemifusion lipidic intermediate. We named this superfamily FUSEXINS: FUSion proteins essential for sexual reproduction and EXoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells and somatic cell fusion (Valansi et al., 2017 ).
