Membrane fusion is essential for many physiological and pathological processes: infection of enveloped viruses, fertilization, intracellular trafficking of vesicles and in some cases of organogenesis. These processes are mediated by specific proteins called fusogens. Fusexins are fusogens that are essential for sexual reproduction and exoplasmic merger of plasma membranes in protists, plants, invertebrates and class II enveloped viruses. The main goal of my project is to characterize the least understood class of membrane fusion: cell-cell fusion processes within the phylum Chordata mediated by proteins from the Fusexin superfamily. I will evaluate whether proteins from mouse gametes with predicted structural similarities to Fusexins fulfill the two criteria to be considered fusogens (i.e. necessity and sufficiency for membrane fusion). In parallel, I will elucidate the mechanisms of action of the amphioxus Br-FF-1 proteins, the first Fusexins described in Chordata. The project involves both high risk and feasible objectives, employing assays that are well established in the Podbilewicz lab as well as the development of new techniques (including in vitro fertilization assays, fusion in heterologous cells, pseudotyping of viruses and virus-cell infection). I expect that results obtained will shed light on the evolution of the Fusexin superfamily that maps to the beginning of the eukaryotic cells and enveloped viruses, and thoroughly characterize the mechanisms of action of cell-cell fusogens. My proposal has the potential to lead to breakthroughs in understanding and manipulating membrane fusion in sexual reproduction, organ development, diseases and tissue repair.