IZUMO1 induces multinucleation of BHK cells. (A) Cells were transfected with either pCI::myrGFP::H2B-RFP (myrGFP), pCI::GCS1/HAP-V5::H2B-RFP, pCI::IZUMO1-V5:H2B-RFP, or pCI::JUNO::H2B-RFP vectors. JUNO was co-transfected with a plasmid for myrGFP. Immunofluorescence was performed with anti-V5 antibodies for IZUMO1 and GCS1/HAP2, while the myrGFP signal is shown for JUNO and vector control (gray). Arrows show cells with more than one nucleus (magenta). Scale bars, 20 µm. (B) The percentage of multinucleation was defined as the ratio between the nuclei in multinucleated cells (NuM) and the total number of nuclei in multinucleated and fluorescent cells in contact that did not fuse (NuC), as follows: % of multinucleation = (NuM/[NuM + NuC]) × 100 (see Table S1). We show individual data and means ± SEM of four independent experiments. The total number of nuclei counted was: myrGFP, 3,846; GCS1/HAP2, 3,301; IZUMO1, 3,353; JUNO, 3,873. The distribution of the different mononucleated and multinucleated cells counted can be found in Table S1. Comparisons were made with one-way ANOVA followed by Dunett’s test against the empty vector. **P < 0.01, ***P < 0.001. (C) Scheme of experimental design. (D) Z-Series of a tetranucleate IZUMO-V5 cell (white arrow). After immunofluorescence using anti-V5 antibody, the syncytium was analyzed by spinning disk confocal microscopy. DAPI-stained nuclei (blue), IZUMO1-V5 (gray), and H2B-RFP (magenta) are shown. The intensity of the nuclear marker differed between nuclei (white arrow), suggesting a recent fusion between cells expressing different levels of H2B-RFP. Numbers on the bottom left side of each panel are optical sections in micrometers. Scale bar, 20 µm. See also Fig. S1 C and Video 1.