Hemifusion in HAP2-mediated fusion is bilateral, and Fusexins use divergent mechanisms. (A) Cartoon illustrates the hemifusion assay in which one cell type, labeled with two fluorescent probes, acts as the “donor” cell and the unlabeled cell acts as “acceptor.” Because of internalization of fluorescent lipid from the plasma membrane, by the time we score fusion, this probe mostly labels intracellular membranes. (B) Fluorescence microscopy images of AtHAP2-transfected cells labeled with both cell tracker (green, content probe) and Vybrant DiI (red, membrane probe) coplated with unlabeled AtHAP2-transfected cells. (top) Green cell tracker, DiI (red), and Hoechst 33342 (blue). (bottom) Green cell tracker and Hoechst 33342 (blue). Hemifusion event is detected as an appearance of a cell (marked by arrows) that acquired only membrane probe apparently from an adjacent double-labeled cell (arrowheads). Bar, 20 µm. (C) Hemifusion extents quantified as the ratio between numbers of cells labeled only with membrane probe and numbers of cells labeled with both membrane and content probes. The results are means ± SEM (n = 3). (D) Viral class II trimeric fusion proteins (viral Fusexins) have a unilateral fusion mechanism, and the Fusexin is present only in the virus’ envelope or in one cell during cell–cell fusion. (E) Somatic cellular Fusexins (e.g., EFF-1 and AFF-1; on green cells) use a bilateral homotypic mechanism, and the model proposes Fusexin has to be in both cells, forming trans-trimeric complexes (Podbilewicz et al., 2006; Pérez-Vargas et al., 2014). Our results suggest a mechanistic model in which sperm AtHAP2 fuses animal cells using a homotypic design (bilateral; pink cells). HAP2 and EFF-1 can fuse cells in trans using a heterotypic mechanism. (F) We hypothesize that the egg of A. thaliana (the central cell too) expresses an unidentified Fusexin that interacts with sperm HAP2.