Structural Snapshots of Eff-1 Mediated Membrane Fusion

Abstract

The mechanism of Eukaryotic cell-cell fusion remains unknown despite its relevance to various developmental pathways. This well regulated process was expected to require complex interaction systems and multiprotein machineries. The discovery of the C. elegans EFF-1 protein as a single and direct fusing agent has challenged this concept. X-ray crystallography revealed that the ectodomain of EFF-1 is structurally homologous to class-II virus membrane fusion proteins. In these viruses, acidification drives a change in the oligomerization state of the fusion protein and exposes a hydrophobic loop. Both events concomitantly target and merge the viral membrane to the cellular membrane. In contrast, EFF-1 needs to be located in both fusing membranes and is proposed to fuse without a hydrophobic targeting loop. In consequence, several opposed hypotheses explaining how EFF-1 fuses cells coexist, whether based or not on the class-II viral membrane fusion protein paradigm. To clarify the mechanism, EFF-1 driven fusion has to be studied in a lipid environment. We reconstituted recombinant EFF-1 produced in Drosophila Schneider 2 cells into nanodiscs and liposomes and immunoisolated EFF-1 enriched extracellular vesicles. A combination of cryo-electron microscopy (single particle, tomography and subtomogram averaging) and spectroscopic techniques (dynamic light scattering) allowed us to assess the EFF-1 oligomerization state, its local concentration and its spatial organization; and to correlate it to its activity requirements. Our data suggests that EFF-1 mediated membrane fusion deviates from the mechanism described for class-II viral fusion proteins. Here, we present EFF-1 fusogen in its natural milieu at near nanometer resolution, deepening structural signatures of a powerful architecture adapted to work homotypically or heterotypically.